Ultrafiltration membrane, device, bioartificial organ, and related methods

ABSTRACT

The present invention relates to ultrafiltration. In particular, the present invention provides nanoporous membranes having pores for generating in vitro and in vivo ultrafiltrate, devices and bioartificial organs utilizing such nanoporous membranes, and related methods (e.g., diagnostic methods, research methods, drug screening). The present invention further provides nanoporous membranes configured to avoid protein fouling with, for example, a polyethylene glycol surface coating.

RELATED METHODS

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/879,744, filed Jan. 10, 2007, which is hereinincorporated by reference in its entirety.

This invention was made with government support under DK 50539 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to ultrafiltration. In particular, thepresent invention provides nanoporous membranes having pores forgenerating in vitro and in vivo ultrafiltrate, devices and bioartificialorgans utilizing such nanoporous membranes, and related methods (e.g.,diagnostic methods, research methods, drug screening). The presentinvention further provides nanoporous membranes configured to avoidprotein fouling.

BACKGROUND OF THE INVENTION

Renal failure affects approximately 300,000 Americans and an unknownnumber of patients worldwide. Treatment methods of kidney failurecurrently include organ transplantation and dialysis. Organtransplantation involves a kidney from a cadaver or a living donorimplanted in the anterior abdominal wall or the peritoneum of thepatient with kidney failure, and the formation of vascular and urinaryconduits. Alternatively, two types of dialysis are available:hemodialysis, where the patient's blood is passed against a synthetic orsemisynthetic membrane and diffusive transport of toxins occurs into abath of dialysate on the other side of the membrane, and peritonealdialysis, wherein the patient's parietal peritoneal epithelium performsthe function of the dialysis membrane. Both dialysis methods areperformed at scheduled periods of time. All of these treatments areseverely limited; organ transplantation is limited by a shortage ofdonor organs, and dialysis is limited by severe morbidity and mortality.There is evidence that the use of slow continuous ultrafiltrationprovides benefits when compared with the use of intermittenthemodialysis currently available. There are also components of abioartificial kidney under development, which may replace some of theendocrine and metabolic functions of the kidney not replaced inhemodialysis.

The replacement of renal function in persons with renal failure bydialysis is dependent on the ability to filter out waste products whilepreserving metabolically costly proteins, peptides, and cells. In bothforms of dialysis, small molecules diffuse from an area of higherconcentration (blood) to an area of lower concentration (dialysate),which are separated either by a membrane of cells (the peritoneallining) in the case of peritoneal dialysis, or a synthetic membrane inthe case of hemodialysis. Transport of a molecule from one fluid to theother is proportional to the difference in concentrations of themolecule in the two fluids and is approximately inversely proportionalto the molecular size, up to sizes excluded by the membrane. Thussmaller molecules are extracted from the blood more quickly than largerones. In the native kidney, this is accomplished by a structure calledthe glomerulus. Blood under arterial pressure enters the glomerularcapillary, and water and small solutes are forced through a specializedtissue structure comprised of the cells and connective tissue of theglomerular capillary tuft. The cellular and molecular structure of theglomerulus imposes constraints based on molecular size and molecularcharge. Molecules meeting certain size and charge constraints aredragged with the fluid and are transported at a rate directlyproportional to the rate of fluid flow. For very small molecules, suchas urea, clearance by either method is similar. For very large molecule,such as antibodies, the blockade to passage is similar. For molecules inbetween, such as β2-microglobulin, convective transport viaultrafiltration may be far more efficient than diffusive clearancethrough dialysis. β2-microglobulin was selected as an exemplary moleculeprecisely because it accumulates in renal failure and causes toxicity inthe patient, and is not effectively removed by dialysis.

Present hemodialysis requires a bulky hollow-fiber dialyzer that canmeasure over twelve inches in length and two inches in diameter, andthat requires extracorporeal pumps to maintain the blood flow. Such anassembly is not suited to implantation, although wearable externaldevices have been tested. Furthermore, conventional hemodialysisrequires a supply of purified sterile nonpyrogenic water with a balancedelectrolyte composition, at flow rates of 400-800 ml/min, which isclearly unsuitable for portable or implantable use. Furthermore, theideal permselectivity of a dialysis membrane is far from settled, withactive research into the relative importance of electrostatic chargeversus steric exclusion. Still further, conventional synthetic orsemisynthetic membranes have a limited service life due to proteinfouling and blood clotting.

What is needed is a filtration membrane which more closely reproducesthe filtration functions of the native kidney, both in adoptingconvective transport of solutes across the membrane and in requiringonly modest transmembrane pressures to effect hemofiltration. What isalso needed are filtration membranes configured to prevent or decreaseprotein fouling, resulting in an increased service life.

SUMMARY OF THE INVENTION

The present invention relates to ultrafiltration. In particular, thepresent invention provides nanoporous membranes having pores forgenerating in vitro and in vivo ultrafiltrate, devices and bioartificialorgans utilizing such nanoporous membranes, and related methods (e.g.,diagnostic methods, research methods, drug screening). The presentinvention further provides nanoporous membranes configured to avoidprotein fouling with, for example, a surface coating such as, forexample, polyethylene glycol, oligosaccharide surfactant polymers,heparin, and hyaluronan. The present invention is not limited to aparticular type of membrane material. In some embodiments, the materialof the membrane is, for example, silicon, polysilicon, silicon carbide,ultrananocrystalline diamond, diamond-like-carbond (DLC), silicondioxide, PMMA, SU-8, PTFE, titanium, silica, silicon nitride,polytetrafluorethylene, polymethylmethacrylate, polystyrene, and/orsilicone. In some embodiments, the membranes are associated with sensorsconfigured to monitor, for example, filtration parameters. Examples ofsuch sensors include, but are not limited to, pressure sensorsconfigured to monitor transmembrane pressure, protein sensors configuredto monitor protein leakage/membrane breakdown, optical blood sensorsconfigured to monitor membrane rupture, and urea sensors configured tomonitor urea clearance.

It is not obvious to those skilled in the art that a protein-freeultrafiltrate generated by the devices of the present invention may bein itself valuable and useful for ends other than the removal of toxinsin blood filtering applications. For example, the ultrafiltrationdevices of the present invention also find use in diagnosticapplications. For example, the devices provides a means for selectivelyscreening out undesired molecules (e.g., proteins) within fluids, suchthat a particular analyte to be analyzed (e.g., small molecules such asglucose, lactic acid, electrolytes, ions, including, but not limited to,potassium, sodium, calcium, chloride, oxygen, and carbon dioxide) in theabsence of interfering molecules. Present electrochemical sensors forglucose measurement are severely hampered by protein fouling of thesensor, and great effort is devoted to the invention of foulingretardants to prolong sensor life. An ultrafiltrate substantially freeof proteins, but still containing smaller constituents of blood,including but not limited to sodium, potassium, chloride, glucose,provides a solution to assay for glucose concentration without proteinfouling. Thus, the present invention further provides systems for use inthe analysis of small molecule, including, but not limited to thoselisted above. Furthermore, as the intracellular aqueous milieu differsfrom extracellular fluid, the separate testing of whole blood and aprotein and cell-free ultrafiltrate for electrolyte compositions,magnetic susceptance, optical, infrared, or magnetic resonancespectroscopy, and other physical properties of matter, provides detailedinformation regarding the cellular composition of the blood.

Furthermore, it is not obvious to those skilled in the art that aprotein and cell free ultrafiltrate of blood so generated may be initself valuable and useful for ends other than the removal of toxins andthe measurement of the constituents of blood. The constituents of bloodnecessary for at least temporary support of a metabolically active cellare small in molecular size (including but not limited to oxygen,glucose, insulin, triiodothyronine, and retinoic acid, for example)while those immune mediators responsible for rejection of an allograftor xenograft are large in molecular size, such as antibodies, orcomponents of the complement cascade, or reside in cell membranes, suchas the major histocompatibility complexes. Thus a stream ofultrafiltrate of blood may be used to supply nutrients and carry awaywastes by an efficient convective transport process, rather than by lessefficient diffusive transport. This is directly applicable to anygeneralized cell population considered for transplantation, includingbut not limited to islet cell transplantation, liver celltransplantation, kidney cell transplantation, and in general transplantof any allo- or xeno-geneic cell type.

The ultrafiltration devices of the present invention also providebioreactors for the growth of cells or tissues. In some suchembodiments, the cells or tissues are grown with a chamber of the devicesuch that the media in which the cells or tissues is bathed isselectively screened by the membranes of the device.

The present invention also provides bioartificial organs for in vivo orextracorporeal uses. In some embodiments, the bioartificial organscomprise cells attached to or associated with a surface. In some suchembodiments, the surface is modified to control the biological activityof the attached or associated cells. In some preferred embodiments, thesurface is a membrane of the present invention, having pores, asdescribed herein. However, the present invention is not limited to theuse of surfaces that comprise the membranes of the present invention.

In some embodiments, the present invention provides systems, methods anddevices that utilize a defined pore shape and structure which mayincorporate electrodes or other devices, chemicals, and treatmentswithin or around a pore structure to control charge and/or sizeselectivity of the pore. The present invention also provides systems andmethods of using such pores to produce an ultrafiltrate; in particular,such methods are used to produce an ultrafiltrate of plasma, therebyaccomplishing hemofiltration and/or hemodialysis.

For example, in some embodiments, the present invention provides amembrane comprising nanofabricated pores, where each pore comprises apore structure of defined dimensions and structure, and density. Infurther embodiments, at least one pore of the membrane and/or optionallyat least a portion of the membrane surface comprises at least onesurface treatment. Surface treatments include but are not limited totreatments that limit protein adsorption, treatments that alter orconfer surface charge and surface free energy and treatments thatpromote adhesion of specific cell types. In other embodiments, at leastone pore of the membrane comprises at least one electrode positioned onor near the membrane and/or pore such that an electric field isgenerated in or near the nanofabricated pore. In yet other embodiments,at least one pore of the membrane comprises any combination of a surfacetreatment, or any combination of a surface treatment and at least oneelectrode. Surface treatments and/or electric fields function to effectrestriction of size and electrostatic charge of solutes that may bepassed through such pores.

In other embodiments, the present invention provides an ultrafiltrationsystem comprising: 1) a membrane comprising nanofabricated pores asdescribed above; 2) an electrode or other device, technique, ormodification to generate an electric field positioned on or near themembrane and/or pore such that an electric field is generated in or nearthe nanofabricated pores; 3) a housing containing the membrane and theelectrode; and a fluid delivery passageway with a first end and a secondend, said first end positioned outside of the housing, the second endpositioned to deliver fluid across the membrane. In further embodiments,the system further comprises a membrane comprising nanofabricated poresas described, wherein the membrane also comprises a surface treatment ofat least one pore and/or of the membrane, wherein the surface treatmentfunctions to promote or retard attachment of specific cells andproteins.

In preferred embodiments, the system is configured to receive anddeliver blood or plasma directly or indirectly from a subject'svasculature. In some embodiments, the housing is very small, allowingthe system to be maintained on or in a subject. For example, in somepreferred embodiments, the housing is made of or coated in abiocompatible material and is implanted into a subject to providecontinuous hemofiltration and/or hemodialysis. In some embodiments, thesystem is attached to one or more additional devices that process,store, or otherwise manipulate a biological fluid and/or collect andanalyze data.

In some embodiments, the system further comprises a pump configured topass fluid through the fluid delivery passageway. In yet otherembodiments, the system further comprises an actuator (e.g., a nanoscaleactuator) that decreases protein fouling of the pores during fluidprocessing.

The present invention also provides methods of filtering a biologicalfluid. For example, in some embodiments, the present invention providesa method having the steps of, 1) providing a biological fluid (e.g.,from a subject) and an ultrafiltration system (e.g., as described above,or elsewhere herein); 2) transferring the biological fluid into theultrafiltration system (e.g., into the first end of the fluidpassageway); 3) passing the fluid across a membrane to generatedfiltered fluid; and, in some embodiments, 4) transferring the filteredfluid to a subject. In some preferred embodiments, the filtered fluidthat is generated is substantially free of proteins. Thus, in someembodiments, the method produces hemofiltered and/or hemodialyzed fluid.

In some preferred methods, an electric field is provided in or around atleast one nanofabricated pore in the membrane. In some embodiments, theelectric field is produced under conditions such that the pores providea charge and/or size selective barrier to proteins. In some embodiments,the electric field is produced under conditions such that proteinfouling is reduced in the pores.

In some embodiments, the present invention provides an ultrafiltrationsystem comprising: a) a membrane comprising micromachined pores having alength and a width, said length being less than 500 microns (e.g., lessthan 200, less than 100, less than 50, less than 20, less than 10, etc.microns) and said width being less than 500 nanometers (e.g., less than200, less than 100, less than 50, less than 20, less than 10, . . .nanometers), wherein the ratio of said length to said width is at least2:1 (e.g., 3:1, 4:1, 5:1, 8:1, 10:1, . . . etc.); a housing containingsaid membrane; and a fluid delivery passageway with a first end and asecond end, said first end positioned outside of said housing, saidsecond end positioned to delivery fluid across said membrane. Inpreferred embodiments, the housing comprises a biocompatible coatingthat permits the system to be used in vivo. In some embodiments, thesystem further comprises one or more electrodes positioned on or nearsaid membrane such that an electric field is generated in or near saidpores. In some embodiments, the housing has a length and a width, saidlength of said housing being less than 500 millimeters (e.g., less than400, 300, 200, 100, . . . ) and said width of said housing being lessthan 500 millimeters (e.g., less than 400, 300, 200, 100, . . . ).

The present invention further provides an ultrafiltration systemcomprising a membrane comprising a plurality of micromachined pores,wherein the length (the longest dimension) of each of said plurality ofmicromachined pores differs from the length from the other micromachinedpores by no more than 30% (e.g., 20%, 10%, 5%, . . . ). In someembodiments, the width (the shortest dimension) of each of the pluralityof micromachined pores differs from the shortest dimension of the othermicromachined pores by no more than 30% (e.g., 20%, 10%, 5%, . . . ).

The present invention further provides an ultrafiltration systemcomprising a plurality of membranes, wherein each of the membranescomprises a plurality of micromachined pores, wherein the shortestdimension of each of the plurality of micromachined pores differs fromthe shortest dimension of the other micromachined pores by not more than30% (e.g., 20%, 10%, 5%, . . . ).

The present invention also provides an implantable ultrafiltrationdevice comprising: a membrane comprising micromachined pores configuredto permit ultrafiltration of blood under systolic blood pressure (e.g.,without the use of a pump); a biocompatible housing containing saidmembrane; and a fluid delivery passageway with a first end and a secondend, said first end positioned outside of said housing, said second endpositioned to delivery fluid across said membrane.

The present invention further provides a diagnostic ultrafiltrationdevice comprising a any of the above membranes; a housing containingsaid membrane; a fluid delivery passageway with a first end and a secondend, said first end positioned outside of said housing, said second endpositioned to delivery fluid across said membrane and into a chamberenclosed by said housing; and a sensor contained in said chamber, saidsensor configure to detect an analyte (e.g., glucose, a pathogen, aportion of a pathogen, etc.).

The present invention also provides a bioartificial ultrafiltrationdevice, comprising: a housing; an inlet port passing through saidhousing, said inlet port configured to receive a biological fluid; anoutlet port passing through said housing, said outlet port configured toreturn a biological fluid to a subject; a membrane contained in saidhousing, said membrane comprising micromachined pores (e.g., anymembrane disclosed herein); and a population of cells attached to saidmembrane. In preferred embodiments, the housing is of a size and is madeof a biocompatible material to allow in vivo use. In some embodiments,the device further comprises one or more electrodes positioned on ornear said membrane such that an electric field is generated in or nearsaid pores. In some embodiments, the population of cells comprises renalproximal tubule cells. In some embodiments, a membrane prevents passageof cells or components of cells, as well as subcellular components, intosaid outlet port or into particular chambers of the device.

The present invention further provides a bioartificial ultrafiltrationdevice, comprising: a housing; an inlet port passing through saidhousing, said inlet port configured to receive a biological fluid, anoutlet port passing through said housing, said outlet port configured toreturn a biological fluid to a subject, a textured surface contained insaid housing, said textured surface configured to support theattachment, growth, normal biological function (e.g., normal proteinexpression), or differentiation of kidney tissue; and a population ofcells attached to said membrane. In some embodiments, the texturedsurface comprises a silicon surface (e.g., silicon or polysilicon). Insome preferred embodiments, the silicon surface comprises asingle-crystal silicon surface. In some embodiments, the surface iscoated with extracellular matrix proteins. In some embodiments, thecells comprise renal tubule cells, pancreatic cells, hepatic cells,thyroid cells, adrenal cells, parathyroid cells, pituitary cells,hypothalamic cells, gonadal cells, prokaryotic cells, duodenal cells,other intestinal cells, gastric cells, muscle cells, fibroblast cells,and endothelial cells. In preferred embodiments, the surface isconfigured such that the renal tubule cells express tight junctionproteins. In some preferred embodiments, the surface is prepared bygenerating an oxide layer, followed by deposition of a polysilicon film.

In some embodiments, the membranes of the present invention do notrequire blood pumps to operate, and as such, simplifies wearable and/orimplantable dialysis. In some embodiments, the present inventionprovides membranes for ultrafiltration with well-defined, slit-shapedpores. In some embodiments, the membranes are designed and assembledusing silicon bulk and surface micromachining techniques. In someembodiments, the membranes are further surface modified with poly(ethylene glycol) (PEG). Experiment conducted during the development ofthe present invention demonstrated that hydraulic permeability of suchmembranes was stable despite perfusion with albumin, showing thatmembrane fouling by BSA, or the Ficoll polymer, did not occur.Conventional polymer dialyzers are estimated to have a mean porediameter around 30 angstrom units (Å), with pore size distributionsencompassing a range possibly double that (see, e.g., Ronco C, et al.,IJAO 24:726-735 (2001); incorporated herein by reference in itsentirety). Hydraulic permeability calculations for the silicon membraneprovided by particular examples of the present invention estimate a poresize of 67 Å by 45 microns. Despite a mean pore size more than twicethat of the polymer membrane in these specific examples, more stringentsize exclusion was observed. A doubling in mean pore size showed afourfold improvement in specific hydraulic permeability (hydraulicpermeability normalized to membrane porosity).

In certain embodiments, the present invention provides membranescomprising a plurality of nanofabricated pores. The present invention isnot limited to a particular type, kind or size of membrane comprising aplurality of nanofabricated pores. In some embodiments, the membrane hasa surface coating of polyethylene glycol. In some embodiments, theplurality of nanofabricated pores have a width less than 100 nanometer(e.g., less than 50, less than 20 nanometers, etc.). In someembodiments, the distance (e.g., average distance) between each of theplurality of nanofabricated pores is less than 500 nanometers (e.g.,less than 50 nanometers, 100 nanometers, 150 nanometers, 200nanometers). In some embodiments, the length of the nanofabricated poresis less than 200 μm (e.g., less than 100 μm, 50 μm, 40 μm, 30 μm, 10μm). In some embodiments, the plurality of nanofabricated pores have aslit shape.

In certain embodiments, the present invention provides anultrafiltration system comprising a) a membrane comprising a pluralityof nanofabricated pores; b) a housing containing the membrane; and c) afluid delivery passageway with a first end and a second end, the firstend positioned outside of the housing, the second end positioned todelivery fluid across the membrane.

In some embodiments, the housing comprises a coating, the coating beingbiocompatible for in vivo use. In some embodiments, the housing isconfigured for implantation into an animal (e.g., a human). In someembodiments, the housing has a length and a width, the length of thehousing being less than 300 millimeters (e.g., 300 millimeters, 250millimeters, 200 millimeters, 175 millimeters, 100 millimeters) and thewidth of the housing being less than 300 millimeters (e.g., 300millimeters, 250 millimeters, 200 millimeters, 175 millimeters, 100millimeters).

In some embodiments, the system further comprises one or more electrodespositioned on or near the membrane such that an electric field isgenerated in or near the pores.

In some embodiments, the system further comprises one or more electrodespositioned on or near the membrane such that an electric field isgenerated in or near the pores.

In certain embodiments, the present invention provides methods offiltering a biological fluid comprising a) providing i) a biologicalfluid; and ii) an ultrafiltration system comprising a) a membranecomprising a plurality of nanofabricated pores having a width less than100 nanometers (e.g., less than 20 nanometers); the membrane optionallyhaving a surface coating of polyethylene glycol; b) a housing containingthe membrane; and c) a fluid delivery passageway with a first end and asecond end, the first end positioned outside of the housing, the secondend positioned to deliver fluid across the membrane; and b) transferringthe biological fluid into the first end of the delivery passageway; andc) passing the fluid across the membrane to generated filtered fluid. Insome embodiments, the method further comprises the step of d)transferring the filtered fluid into a subject. In some embodiments, thefiltered fluid is substantially free of proteins. In some embodiments,the filtered fluid comprises hemofiltered fluid.

In certain embodiments, the present invention provides an implantableultrafiltration device comprising a) a membrane comprising a pluralityof nanofabricated pores having a width less than 100 nanometers (e.g.,less than 20 nanometers); the membrane optionally having a surfacecoating of polyethylene glycol; b) a biocompatible housing containingthe membrane; and c) a fluid delivery passageway with a first end and asecond end, the first end positioned outside of the housing, the secondend positioned to delivery fluid across the membrane. In someembodiments, the housing comprises a coating, the coating beingbiocompatible for in vivo use. In some embodiments, the device furthercomprises a population of cells attached to the membrane. In someembodiments, the cells are selected from the group consisting of renaltubule cells, pancreatic cells, hepatic cells, thyroid cells, adrenalcells, parathyroid cells, pituitary cells, hypothalamic cells, gonadalcells, prokaryotic cells, duodenal cells, gastric cells, intestinalcells, muscle cells, fibroblast cells, and endothelial cells. In someembodiments, the housing has physical dimensions that permit the deviceto be used in a human subject, in vivo.

In certain embodiments, the present invention provides methods forcreating an implantable ultrafiltration device comprising imprinting aplurality of nanofabricated pores having a width of less than 20nanometers onto the surface of a membrane, wherein said nanofabricatedpores are positioned in the center of said membrane surface. In someembodiments, the imprinting is accomplished with nanolithography. Insome embodiments, the membrane has a surface coating selected from thegroup consisting of polyethylene glycol, oligosaccharide surfactantpolymers, heparin, and hyaluronan. In some embodiments, the membrane hasa porosity below 2%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic description of process flow for the fabricationof nanoporous membranes showing wafer cross-sections.

FIG. 2 shows a graph of hydraulic permeability of nanofabricatedmembranes of the present invention, with hydraulic permeabilites of twocommercial polymer dialysis membranes (Baxter CT110 and Fresenius F-80)plotted for comparison.

FIG. 3 shows an extracorporeal hemofiltration device in some embodimentsof the present invention.

FIG. 4 shows a continuous analyte sensor in some embodiments of thepresent invention.

FIG. 5 shows a bioartificial organ in some embodiments of the presentinvention.

FIG. 6 shows a bioartificial organ in some embodiments of the presentinvention.

FIG. 7 shows a custom-built test apparatus for pressure-driven flowcharacterization experiments.

FIG. 8, taken from Sharma et al., 2004 Langmuir 20(2), shows theresistance of PEG coatings to protein fouling. LEFT—Fluorescenceintensity for unmodified and PEG-modified surfaces over a period of 4weeks. Samples were incubated in FITC-BSA (0.5 mg/mL in PBS, pH 7.4, 37°C.) for 1 hour. CENTER—Adhesion of fibroblasts to silicon andPEG-modified silicon substrates: (A) control (silicon); (B) PEG-modifiedsilicon (O week incubation period); (C) PEG-modified silicon (after4-week incubation period). RIGHT—Variation in protein adsorbed on PEGthin films of various grafting densities (ellipsomeric measurements0.Solid lines show the fit for the expotential decay model(y=y_(o)+ae^(−bx)).

FIG. 9 shows silicon membranes coated with PEG have increased plasmarecalcification time in comparison to silicon membranes lacking PEGcoating and glass slides.

FIG. 10 shows a nanoporous membrane of an embodiment of the presentinvention. Fabrication of nanoporous membranes (A-I) depicted via wafercross-sections (not to scale). The critical pore dimension (SiO2thickness in C) can be controlled between 5-50 nm just by changingoxidation parameters. SEM images of nanoporous membrane showing top view(J) and cross-section (K). Scatter plot (L) shows across-wafer oxidethickness uniformity variation of <1% (maximum 3%) from 25 differentoxidation runs.

FIG. 11A shows sieving coefficients of candidate proteins throughnanoporous membrane (30 nm-wide slit pores) showing somepermselectivity. FIG. 11B shows sieving coefficients (σ) of Ficoll 70and BSA compared to theoretical prediction based on steric hindrance. λrefers to the ratio of molecule diameter to nanopore size. σBSA=0.24 foran 8 nm pore.

FIG. 12 shows the hydraulic permeability of a nanoporous membrane of thepresent invention.

FIG. 13 shows the permselectivity of a nanoporous membrane of thepresent invention.

FIG. 14 Left shows human renal tubule epithelial cell growth on siliconnanoporous membranes. Renal tubular epithelial cells stained for a tightjunction protein, ZO-1 (green), a ciliary protein, acetylated tubulin(red), a brush border enzyme, γ-glutamyl transferase (not shown), and anuclear stain (blue). FIG. 14 RIGHT shows transepithelial resistance ofrenal tubule epithelial cells on silicon nanoporous membranes iscomparable to conventional polyester controls (dashed line) for 0-96hours in culture.

FIG. 15 shows an exemplary membrane manufacture method in someembodiments of the present invention.

FIG. 16 shows an exemplary membrane manufacture method in someembodiments of the present invention.

FIG. 17 shows fabrication of silicon nanoporous membranes using MEMS andnanoimprint lithography technology. Cross-sectional schematics depictthe wafer at various stages of fabrication. These drawings are not drawnto scale in order to show the details of the cross-sectional flow.

FIG. 18 shows the stress fields and fracture probability of threemembrane designs. Design #A shows a baseline solid membrane without anypores, while Design #B presents a membrane design having 2.9% porositywherein the pores are distributed all over the membrane including theedge and center regions. The corresponding fracture probabilities (˜0%for Design #A and 55% for Design #B) indicate that current membranedesigns are fragile and fracture slightly above 3 psi, while the solidmembranes survive 100 psi transmembrane pressures. In contrast, poredistribution in Design #C has been optimized to avoid membrane edges andthe center region, which results in a very low fracture probability(˜0%).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases as used herein are defined below:

As used herein, the term “filtration” refers to a process of separatingparticulate matter from a fluid, such as air or a liquid, by passing thefluid carrier through a medium that will not pass the particulates.

As used herein, the term “ultrafiltration” refers to subjecting a fluidto filtration, where the filtered material is very small; typically, thefluid comprises colloidal, dissolved solutes or very fine solidmaterials, and the filter is a microporous, nanoporous, orsemi-permeable medium. A typical medium is a membrane. The fluid to befiltered is referred to as the “feed fluid.” During ultrafiltration, thefeed fluid is separated into a “permeate” or “filtrate” or“ultrafiltrate,” which has been filtered through the medium, and a“retentate,” which is that part of the feed fluid which did not getfiltered through the medium, or which is retained by the medium.

As used herein, the term “dialysis” refers to a form of filtration, or aprocess of selective diffusion through a membrane; it is typically usedto separate low-molecular weight solutes that diffuse through themembrane from the colloidal and high-molecular weight solutes which donot. In some embodiments, a feed of fluid is passed over a semipermeablemembrane, and a feed of dialysate is passed over the other side of thatmembrane; the membrane is wetted by one or both solvents, and then thereis diffusive transport of dissolved solutes between the fluids. Thecomposition of one fluid, the dialysate, is used to deplete thecomposition of the other fluid, the feed fluid, of some molecule ormolecules.

As used herein, the term “dialysate” is used to refer to the fluid intowhich low-molecular weight solutes diffuse through a membrane fromanother fluid (typically, the feed fluid) initially containing thesesolutes.

As used herein, the term “free of” refers to fluids of mixtures thathave had one or more components (e.g., protein components) removed.“Substantially free of” fluids or mixtures are at least 50% free,preferably at least 75% free, and more preferably at least 90% free froma component with which they are otherwise naturally associated. Forexample, a fluid that is “substantially free of protein” is a fluid thathas at least 50% or less of the protein content of an unfiltered orunpurified fluid.

As used herein, the term “microelectronics” refers to a branch ofelectronics that deals with the miniaturization of electroniccomponents.

As used herein, the term “microchip” refers to another term formicrosized electronic components using integrated circuit technology.

As used herein, the term “microelectromechanical systems” refers todevices that involve integrated microdevices or systems, combined withelectrical and mechanical components, produced usingmicroelectronics-compatible batch-processing techniques. These systemsmerge computation with sensing and actuation to perceive the physicalworld at a miniaturized level.

As used herein, the term “MEMS” refers to a mnemonic formicroelectromechanical systems.

As used herein, the term “microfluidics” refers to MEMS devices used forthe movement of fluids or gases to create microscale chemical analysissystems. This technology is becoming widely used in ink-jet printingdevices for increased accuracy and resolution. It is also beinginvestigated for its use in DNA analysis and synthesis where minutequantities of fluid are needed to assess the biochemical makeup of acell or protein.

As used herein, the term “microfabrication” refers to a processingtechniques used to manufacture microelectronics components. Typicaltechniques are deposition, photolithography, etching, and doping.

As used herein, the term “micromachining” refers to mechanical andchemical fabrication processes that were used to form thesemicromechanical parts, such as by etching areas of the silicon substrateaway to leave behind the desired geometries. The development of siliconmicrosensors often required the fabrication of micromechanical parts(e.g., a diaphragm in the case of the pressure sensor and a suspensionbeam for many accelerometers). These micromechanical parts werefabricated by selectively etching areas of the silicon substrate away toleave behind the desired geometries. Hence, the term micromachining cameinto use in the early 1980s. Micromachining designates the mechanicalfabrication processes that were used to form these micromechanicalparts. The successful incorporation of techniques for the selectiveetching of silicon (which were initially investigated in the 1960's and1970's), with advances in microfabrication, provided the processflexibility that was necessary to fashion micromechanical parts fromsilicon and related microelectronics fabrication materials.

As used herein, the term “polysilicon” refers to a polycrystalline formof silicon that is deposited as a thin film. It is used inmicroelectronics for transistors and wiring. In MEMS, polysilicon isusually used as structural material for devices.

As used herein the term “animal” refers to any member of the kingdomAnimalia that includes living things which have cells differing fromplant cells with regard to the absence of a cell wall and chlorophylland the capacity for spontaneous movement. Preferred embodiments of thepresent invention are primarily directed to vertebrate (backbone ornotochord) members of the animal kingdom.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulardiagnostic test or treatment. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animalsand plants, including but not limited to, body fluids such as urine,blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, aswell as solid tissue, sap, and nectar. However, these examples are notto be construed as limiting the sample types applicable to the presentinvention.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

DETAILED DESCRIPTION

The kidney is unique in that it is the first organ for which long-termex vivo substitutive therapy has been available and lifesaving. Renalfailure prior to the era of hemodialysis and transplantation resulted incertain death, and this outcome of renal failure is still currentoutside the industrialized world.

In the United States, 452,000 patients were listed as having end-stagerenal disease (ESRD) by the 2005 USRDS database, of whom 324,826 werereceiving maintenance dialysis (see, e.g., U.S. Renal Data System, USRDS2005 Annual Data Report: Atlas of End-Stage Renal Disease in the UnitedStates, National Institutes of Health, National Institute of Diabetesand Digestive and Kidney Diseases, Bethesda, Md. 20002; incorporatedherein by reference in its entirety). The prevalence of ESRD in theUnited States is rising at approximately 8% per year (see, e.g., U.S.Renal Data System, USRDS 2005 Annual Data Report: Atlas of End-StageRenal Disease in the United States, National Institutes of Health,National Institute of Diabetes and Digestive and Kidney Diseases,Bethesda, Md. 20002; Neilson E G et al: The Ad Hoc Committee Report onestimating the future workforce and training requirements fornephrology. JASN 1997; 8:S1-S4; each incorporated herein by reference intheir entireties). The financial cost of dialysis is immense, estimatedat 64,614 USD per hemodialysis patient per year and 47,384 USD perperitoneal dialysis patient per year. In contrast, transplant patientscost an average of 22,142 USD per patient per year (see, e.g., U.S.Renal Data System, USRDS 2005 Annual Data Report: Atlas of End-StageRenal Disease in the United States, National Institutes of Health,National Institute of Diabetes and Digestive and Kidney Diseases,Bethesda, Md. 20002; incorporated herein by reference in its entirety).

The higher cost of maintenance dialysis when compared withtransplantation does not translate into better results; annual mortalityfor patients listed for transplant and awaiting a kidney is 6.3%,compared with only 3.8% for patients listed for transplant who didreceive a kidney. These statistics compare favorably to the 16.7% annualmortality for ESRD patients not listed for transplant (see, e.g., WolfeR A et al: Comparison of mortality in all patients on dialysis, patientson dialysis awaiting transplantation, and recipients of a firstcadaveric transplant. NEJM 1999; 341:1725-1730; incorporated herein byreference in its entirety). Transplantation, despite its advantages interms of cost, morbidity, and mortality, is severely limited by scarcityof donor organs. In 2006, there are over 300,000 patients on dialysisand 72,983 patients on the kidney wait list. Only 4096 renal transplantswere performed in the first quarter of 2006, based on Organ Procurementand Transplantation.

Frequent prolonged dialysis administered at home reduces comorbiditiesand enhances patient lifestyle at significantly reduced cost, but thelabor, risk, and inconvenience of conventional dialysis may limitacceptance outside of a clinical trial environment (see, e.g., PierratosA., et al, Quotidian dialysis—update 2005 Current Opinion in Nephrology& Hypertension. 14:119-24 (2005); Chan C T, et al., KidneyInternational. 61:2235-9 (2002); Lindsay R M, et al., American Journalof Kidney Diseases. 42:24-9 (2003); Heidenheim A P; et al., AJKD.42:36-41 (2003); each incorporated herein by reference in theirentireties). Recent data suggest that convective or mixedconvective-diffusive therapies are associated with enhanced survivalcompared with diffusive therapies alone (see, e.g., Canaud B, et al.,Kidney Int. 69:2087-2093 (2006); Jirka T, et al., Kidney Int 70:1524(2006); each incorporated herein by reference in their entireties).

Prolonged hemodiafiltration or hemofiltration at home improves outcomesin ESRD care. However, technology is not in place to facilitatewidespread adoption of a home hemofiltration program. Hemodialysis andhemofiltration cartridges in present clinical use requiresuperphysiologic pressures for blood flow through hollow fiber membranesand to drive ultrafiltration. These pressures necessitate roller pumpsand a cumbersome extracorporeal circuit, as well as percutaneousvascular access. Achievement of clinically significant ultrafiltrationrates with physiologic pressures (20-50 mmHg) will simplify engineeringof a wearable or implantable dialysis cartridge. The advantages of animplanted dialysis system include, for example, portability, the safetyinherent in elimination of the extracorporeal blood circuit, as well asthe relocation of the transcutaneous access to the dialysate side of thecircuit, possibly further reducing infection and bleeding.

For convective therapies, an estimate of the hydraulic permeabilityneeded to provide adequate small-solute clearance can be obtained bydividing a minimum ultrafiltration rate, e.g. 30 ml/min, by capillaryperfusion pressure, e.g., 30 mm Hg, obtaining a rough estimate of 1ml/min/mmHg. Conventional polymer dialysis membranes haveultrafiltration coefficients (K_(UF)) of approximately 11-85ml/hour/mmHg (0.18-1.41 ml/min/mmHg), in packages providing 1-2 m²membrane area (see, e.g., Canaud B, et al., Kidney Int. 69:2087-2093(2006); incorporated herein by reference in its entirety). Thesehydraulic data do not incorporate the additional pressure head needed tomove blood along the length of the hollow fiber. These pressures, aswell as the package size of the dialyzer, dictate that present systemsare limited to extracorporeal use.

Maximizing hydraulic permeability, or, reciprocally, minimizing membranearea, is a function of membrane pore geometry. Ronco et al have reviewedthe relationship between pore dimension, size dispersion, and K_(UF)(see, e.g., Ronco C, et al., IJAO 24:726-735 (2001); incorporated hereinby reference in its entirety). In particular, the importance of a narrowpore size distribution in maximizing hydraulic permeability whilelimiting albumin leakage is identified. In addition to pore sizedistribution, membrane thickness and pore shape also influence hydraulicpermeability. Nature has repeatedly evolved slit-shaped or plate-shapedstructures where pressure-driven filtration occurs, such as in thepodocyte slit diaphragm, the bills of filter feeding birds such asshovelers and flamingoes, and in the baleen plates of filter feedingwhales. Elongated, slit-shaped pores are predicted to have highhydraulic permeability per area of membrane, up to a theoretic maximumof about a 2.6-fold increase over round pores.

The present invention provides a compact ultrafiltration device andmethods for generating an ultrafiltrate, both of which can be used for avariety of applications, including, but not limited to filtering blood,diagnostic applications, as a bioreactor, in bioartificial organs, etc.The present invention also provides a nano-machined porous structurethat permits individual control of pore size and charge density.

For example, in some embodiments, the present invention provides amembrane comprising a plurality of pores, where the shapes and sizes ofthe pores are highly controlled. In some embodiments, the membranefurther comprises at least one surface treatment. In other embodiments,the membrane further comprises at least one electric field generator,such that an electric field is produced in or around at least one pore;examples of electric field generators include but are not limited toelectrodes. In yet other embodiments, the membrane further comprises atleast one surface treatment and at least one electric field generator,such that an electric field is produced in or around at least one pore;examples of electric field generators include but are not limited toelectrodes. In yet further embodiments, the membrane further comprisesat least one of a pump and an actuator; in yet further embodiments, themembrane further comprises at least one of a surface treatment, anelectric field generator, such that an electric field is produced in oraround at least one pore, a pump, and an actuator.

The present invention also provides a system comprising a compartmentand the porous membrane of the present invention as described above,where the porous membrane is supported within the compartment of thedevice, such that the presence of the membrane separates the compartmentinto two sub-compartments. A housing defines the outer surfaces of thecompartments. A housing may be composed of any desired material. Wherethe system is used on or in a subject, the housing is preferably made ofor coated with a biocompatible material.

Unlike the system of the prior art, the present invention provides asystem that permits complete manipulation of parameters to controlexclusion of molecules with particular properties (e.g., size, molecularcharge, etc.). The prior art systems also do not permit exclusion ofmolecules within tight property parameters (e.g., sharp size, molecularcharge, etc. cut-offs). Furthermore, the prior art systems do notprovide ultrafiltration systems that can be used in vivo underbiological pressures. Such systems would require the use of pumps tofunction under biological pressures, pumps that are too large forpractical in vivo use.

Wearable or implantable dialysis has been repeatedly proposed sincedialysis was first implemented as treatment for end-stage renal disease(see, e.g., Ohlson M, et al., Am. J. Physiol Renal Physiol 279:F84-F91(2000); Stephen R L., et al., Kidney International—Supplement.(8):S125-32, 1978 June; Nissenson A R, et al., Blood Purification.23(4):269-74, 2005; each herein incorporated in their entireties).Miniaturizing the extracorporeal circuit has been challenging in designof pumps that can circulate blood through hollow fiber dialyzers (see,e.g., Nissenson A R, et al., Blood Purification. 23(4):269-74, 2005;herein incorporated by reference in its entirety). In some embodimentsof the present invention, the membranes do not require blood pumps tooperate, and as such, simplifies wearable and/or implantable dialysis.Accordingly, the present invention relates to ultrafiltration. Inparticular, the present invention provides nanoporous membranes havingslit pores for generating in vitro and in vivo ultrafiltrate, devicesand bioartificial organs utilizing such nanoporous membranes, andrelated methods (e.g., diagnostic methods, empirical methods, drugscreening). The present invention further provides nanoporous membranesconfigured to avoid protein fouling with, for example, a polyethyleneglycol surface coating.

I. Membranes

Existing polymer membranes used in dialysis and ultrafiltration areunsuitable for use in implantable bioartificial kidneys, as they havelow hydraulic permeability and result in prohibitively large packagesize. These membranes are typically produced by formed by extrusion andsolvent casting techniques. The geometry and surface chemistry of thepores arise from the chemistry of the polymers and the fluid dynamics ofthe casting process. In general, the hollow-fiber membranes are fairlythick or employ a multilayer scaffold for mechanical support, and have adistribution of pore sizes rather than a regular array of uniform pores.Pores in conventional polymeric membranes tend to be either roughlycylindrical, have a round orifice terminating a larger channel, or havea structure resembling an open-cell sponge.

The wide dispersion in pore sizes within a membrane leads to imperfectretention of molecules larger than the mean pore size of the membrane.This effect is remedied in practice by engineering the mean pore size ofthe membrane to be sufficiently small that negligibly few pores are solarge as to allow passage of a solute above the desired molecular weightcutoff of the membrane. Unfortunately, this approach has the undesiredeffect of reducing the mean pore size in the membrane and thus reducingthe hydraulic permeability of the membrane. The membranes of the presentinvention overcome these problems with narrower pore size distributions,allowing sharper transitions from passage to retention and maximizingthe mean pore size of the membrane (e.g., nanaporous membranes havingslit pores).

In some embodiments, the present invention provides membranes configuredfor filtration of biological fluids (e.g., dialysis). The presentinvention is not limited to a particular type of membrane. In someembodiments, the membrane comprises a plurality of pores, where theshapes and sizes of the pores are highly controlled. In preferredembodiments, the membrane comprises slit pores. In some embodiments, themembrane further comprises at least one surface treatment. In otherembodiments, the membrane further comprises at least one electric fieldgenerator, such that an electric field is produced in or around at leastone pore; examples of electric field generators include but are notlimited to electrodes. In some embodiments, the membrane furthercomprises at least one surface treatment. In some embodiments, themembrane further comprises at least one of a pump and an actuator. Inpreferred embodiments, the membranes of the present invention areconfigured to generate physiological ultrafiltration volumes atcapillary perfusion pressure.

A. Materials

The membranes of the present invention include any membrane materialsuitable for use in filtering biological fluids, wherein the membranescan be associated with nanofabricated pores. Examples of suitablemembrane materials are known in the art and are describe herein.

In some embodiments, the membrane material is synthetic, biological,and/or biocompatible (e.g., for use outside or inside the body).Materials include, but are not limited to, silicon, which isbiocompatible, coated silicon materials; thus, materials include but arenot limited to, silicon, polysilicon, silicon carbide,ultrananocrystalline diamond, diamond-like-carbond (DLC), silicondioxide, PMMA, SU-8, and PTFE. Other possible materials include metals(for example, titanium), ceramics (for example, silica or siliconnitride), and polymers (such as polytetrafluorethylene,polymethylmethacrylate, polystyrenes and silicones).

B. Nanofabricated Pores

A membrane of the present invention comprises at least one pore, wherepore shapes include but are not limited to linear, square, circular,ovoid, elliptical, or other shapes. In some embodiments, the membranecomprises more than one pore, where the pores comprise a single shape orany combination of shapes. In some embodiments, a membrane comprisesmore than one pore, where the pore sizes range from about 10 to about100 microns in any dimension; the dimensions need not be the same in anyparticular pore shape, the pores may comprise a single size or anycombination of sizes. Although it is not necessary to understand themechanism of invention in order to practice it, and although it is notintended that the invention be limited to any particular mechanism, itis contemplated that slit-shaped pores are the preferred structureresponsible for the filtration specificity of the kidney.

Experiments conducted during the course of the present invention showedthat membranes having slit pores retain sufficiently large solutes butprovide improved hydraulic permeability when compared to cylindricalpores. The round shape of pores in conventional polymer membranesprovides a fourth-power dependence of the volumetric flow rate on thepore radius r:

$\begin{matrix}{\frac{Q}{A\;\Delta\; P} = {\frac{N\;\pi\; r^{4}}{8\mspace{14mu}\mu\; L} = \frac{ɛ\; r^{2}}{{8\mspace{14mu}\mu\; L}\;}}} & (1)\end{matrix}$where Q is the volumetric flow rate, ΔP is the hydrostatic pressuredifference across the membrane, A is the membrane area, N is the numberof pores per unit membrane area, μ is the viscosity, L is the length ofthe pore, and is the membrane porosity (pore area per unit membranearea). In contrast, the volumetric flow rate through a slit-shaped poreis given as:

$\begin{matrix}{\frac{Q}{A\;\Delta\; P} = {\frac{{Nwh}^{3}}{12\mspace{14mu}\mu\; L} = \frac{ɛ\; h^{2}}{12\mspace{14mu}\mu\; L}}} & (2)\end{matrix}$where w is the long dimension of the slit, h is the critical width s ofthe slit, and L is again the length of the pore. For membranes that havea critical dimension that is just able to retain albumin(h_(crit)=2r_(crit)), the volumetric flow rate through the membrane withslit-shaped pores will be 2.67 times larger than the flow rate through amembrane with cylindrical pores (assuming comparable porosity, porelength, and pressure driving force). In addition, the membrane withslit-shaped pores provides much less steric hindrance to the transportof important “middle molecules” such as β₂-microglobulin due to theweaker interactions in the slit geometry. For example, a molecule with a10% smaller radius than the critical pore size will have a sievingcoefficient of approximately 0.1 through the slit-shaped pore comparedto a sieving coefficient of 0.01 for the membrane with cylindricalpores. At equivalent volumetric flow rates of 30 ml/min, this differencecorresponds to a 10-fold larger clearance (3 ml/min versus 0.3 ml/min)for the membrane with slit-shaped pores. This difference increases to26.7-fold for membranes with the same porosity, thickness, and pressuredriving force due to the greater hydraulic permeability of the membranewith slit-shaped pores.

The membranes of the present invention having slit pores are not limitedto having a particular slit pore size. In some embodiments, the size foreach slit pore is approximately 40 μm in length (e.g., 0.m μl in lengthto about 100 μm in length, although other lengths are contemplated). Themembranes are not limited to having thereon a particular number of slitpores. In some embodiments, the membrane has approximately 10⁴ slitpores (e.g., 10² slit pores, 10³ slit pores, 10⁴ slit pores, 10⁵ slitpores, etc). In preferred embodiments, the number of slit pores on themembrane is sufficient to allow the membrane to generate physiologicallyultrafiltration volume at capillary perfusion pressure. The membraneshaving slit pores are not limited to a particular porosity. In someembodiments, the porosity of the membranes is approximately 1% (e.g.,0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, etc.).

In some embodiments, the present invention provides a series ofmembranes comprising sparse arrays of monodisperse slit-shaped pores,manufactured, for example, using silicon bulk and surface micromachiningtechniques (see e.g., Fissell W H, et al., J Am Soc Nephrol 2002; 13:602A; incorporated herein by reference in its entirety). In particular, thepresent invention provides membranes for ultrafiltration withwell-defined, slit-shaped pores designed and assembled using siliconbulk and surface micromachining techniques followed by surfacemodification with poly (ethylene glycol) (PEG). In experiments conductedduring the course of the present invention, hydraulic permeability of aprototype membrane was measured before and after 200 hours of perfusionwith bovine serum albumin. Convective and diffusive transport acrossprototype membranes was quantified and compared with a state of the artpolysulfone membrane using Ficoll 70, a polydisperse spherical polymerof epichlorohydrin and sucrose. Hydraulic permeability of unmodifiedprototype membranes exactly matched first-principles predictions forliquid flow. Pore size was further confirmed by observation of Knudsenflow for argon, nitrogen, and carbon dioxide. Hydraulic permeability ofPEG-modified membranes was consistent with slight pore narrowing by thecoating, and was unchanged by extended perfusion with albumin. Themolecular weight at which the sieving coefficient for Ficoll washalf-maximal (0.5) was around 18 kD, compared with less than 10 kD for apolysulfone membrane. As such, the membranes of the present inventionyielded membranes capable of generating physiologically significantultrafiltration volumes at capillary perfusion pressure. The membranesof the present invention are fundamentally enabling for wearable orimplantable renal replacement.

In some embodiments, the sizes of the slit pores are highly uniform. Forexample, in some embodiments, the pores are micromachined such thatthere is less than 20% size variability, more preferably less than 10%size variability between the dimensions of the slit pores. In furtherembodiments, the sizes of the highly uniform slit pores are ofapproximate dimensions that are similar to the size of the glomerularslit diaphragm, or about 2-100 nm by 2-100 microns. In such embodiments,it is contemplated that the pores permit ultrafiltration at in vivopressures (e.g., systolic blood pressure) (e.g., capillary perfusionpressure). Additionally, it is contemplated that such pores permit sizeselective exclusion of undesired molecules within specific sizerestrictions.

Pressure driven (ΔP) flow Q of incompressible fluid of viscosity μthrough a narrow pore or pipe of rectangular cross section w×h andlength L where h<<w is described by:Q=(wh ³/12 μL)ΔP

And thus flow per unit area Q_(A) of pore w×h is given byQ _(A)=(h ²/12 μL)ΔP

Pressure driven (ΔP) flow Q of incompressible fluid of viscosity μthrough a narrow pore or pipe of round cross section of diameter h andlength L is described byQ=[π(h/2)⁴/8 μL]ΔPorQ=(πh ⁴/128 μL)ΔP

And thus flow per unit area Q_(A) of a round pore of area π(h/2)₂ isgiven byQ _(A)=[(h/2)²/8 μL]ΔPorQ _(A)=(h ²/32 μL)ΔP

Thus for a given critical dimension h of a pore, a rectangular crosssection pore with minimum dimension h has a higher hydraulicpermeability per unit area than does a round pore of diameter h, by afactor of 2.6

Factors that determine appropriate pore size and shape include a balancebetween hydraulic permeability and solute permselectivity. It iscontemplated that a slit shape is an optimal shape, although the presentinvention is not limited to slit shapes.

In preferred embodiments, the slit pores are created by micromachining(referred to as “nanofabrication”) techniques. Micromachining is aprocess that includes photolithography, such as that used in thesemiconductor industry, to remove material from, or to add material to,a substrate. These techniques are well known (see, for example, Park, Bet al. (2002) Med Device Technol 13(2): 32-34; Voldman, J et al. (1999)Annu Rev Biomed Eng 1: 401-425; and Wagner, B (1995) Endosc Surg AlliedTechnol 3(4): 204-209; Encyclopedia of Chemical Technology, Kirk-Othmer(1995), Volume 14, pp 677-709; Rierret, R F (1996) Semiconductor DeviceFundamentals (Addison-Wesley); and Van Zant (1997) Microchip Fabrication3^(rd). edition (McGraw-Hill); Petersen, K E (1982) Proceedings of theIEEE 70:420-457; Roy S, and Mehregany M (1999) Introduction to MEMS, inMicroengineering Aerospace Systems (eds: Helvajian H; The AerospacePress; El Segundo, Calif.) pp. 1-28., and U.S. Pat. No. 6,044,981; eachof which are incorporated herein by reference in their entireties).Although not specifically limited, in preferred embodiments, 1 mm×1 mmnanoporous membranes are generated having thereon approximately 10⁴ 40μl-long slit pores, with an overall porosity of approximately 1%. Inpreferred embodiments, the nanoporous membranes of the present inventionresemble the filtration specificity of the kidney, and represent animprovement over existing membranes having non-slit shaped pores (e.g.,the membranes are able to retain sufficiently large solutes but provideimproved hydraulic permeability when compared to cylindrical pores).

C. Additional Components

1. Surface Treatments

In some embodiments, the membrane further comprises at least one surfacetreatment or modification. In some preferred embodiments, the surfacetreatment or modification promotes attachment of specific animal cellsto the membrane, promotes attachment of desirable proteins, inhibitsundesirable protein deposition on the membrane, or inhibits bloodcoagulation on or in the vicinity of the membrane. Such treatments ormodifications may include but are not limited to patterned orunpatterned adsorption or covalent linkage to the membrane surface ofRGD peptide moieties, integrins, fibronectin, laminin, collagens,oligosaccharides, or polyethylene glycol moieties. Particular cells ormolecules attached to or located at the membrane surface and/or withinthe pores may be used to render the porous membrane more biocompatible,less thrombogenic, or may be used to alter the filtrationcharacteristics of the pores. Furthermore, the cells may be used toprocess or modify the filtrate produced by the membrane. In someembodiments, modification of the pores includes but is not limited tocovalent attachment of peptides or proteins, either alone or selected topromote attachment of cells such as endothelial or epithelial cells.Methods to modify silicon and silicon compounds to promote cellattachment or to retard cell attachment are well known (see, forexample, Whitesides et al. (1996) PNAS 93: 10775-10778 for cellattachment; and Whitesides et al. z91997) Exp Cell Research: 305-313 forpatterned attachment; each herein incorporated by reference in theirentireties).

In some embodiments, the surface of the nanoporous membranes of thepresent invention are modified with polyethylene glycol (PEG) or relatedcompounds (e.g., oligosaccharide surfactant polymer monolayers). Thepresent invention is not limited to a particular method for modifyingnanoporous membrane surfaces with PEG (see, e.g., Papra A, et al.,Langmuir 17:1457-1460 (2001); incorporated herein by reference in itsentirety). Grafting of polyethylene glycol (PEG) monolayer coatings toenhance biofouling resistance of inorganic substrates such as glass andsilicon has been investigated. Two surface modification strategies havebeen established: (1) a multi-step non-aqueous solution synthesis; and(2) a chemical vapor deposition (CVD) method. The CVD route uses aheated stream of ethylene oxide (EtO) with boron trifluoride (BF₃) as acatalyst in a nitrogen carrier gas stream. The surface is firstpretreated with 3-aminopropyltrimethoxysilane to form a uniform surfacecoating with free amine groups. The CVD reaction takes place at the freeamine groups and provides a uniform coating suitable for nanoscalefeatures. The solution based PEG attachment is a multi-step synthesisthat involves the conversion of PEG to PEG-O—SiCl₃ as a reactiveintermediate, which is then coupled with free surface Si—OH groups. Thesurface concentration of the silanol groups is enhanced by apretreatment of the surface with a piranha solution (1:3 H₂O₂:conc.H₂SO₄ v/v). Modification of the nanoporous membrane surfaces with PEGreduced protein fouling (e.g., the PEG coating limited albuminadsorption, and reduced thrombosis initiation) (see, e.g., Sharma andDesai, Journal of Nanoscience and Nanotechnology, 2005, 5(2); Popat andDesai—Biosensors and Bioelectronics, 2004, 19(9); Fissell, et al.,American Journal of Physiology—Renal Physiology, Vol. 293, 2007, p.1209).

2. Electric Field Generators

In preferred embodiments, the membranes have one or more electric fieldgenerators associated with them, such that an electric field is producedin or around the pores. The electric field is used, for example, tocontrol and adjust the relative contributions of electrostatic chargeand steric hindrance across a pore.

In some embodiments, an electric field is created in and around pores ofa membrane by any of several means; this means include, but are notlimited to, electrodes. The electrodes may be located within the pores,or on either side of the pores, or on the surface of the membrane inwhich the pores are fabricated.

The electrodes may be formed by well-known semiconductor processingtechniques from conductive materials, such as pure metals or alloys, orother materials that are metallic conductors. Examples include but arenot limited to aluminum, carbon (such as graphite), cobalt, copper,gallium, gold, indium, iridium, iron, lead, magnesium, mercury (such asamalgam), nickel, niobium, osmium, palladium platinum, rhenium, rhodium,selenium, silicon (such as highly doped polycrystalline silicon),silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc,zirconium, platinum, palladium, iridium, or any combination or alloys ofthese metals; noble metals and their alloys are unreactive in biologicalsystems. The thickness of the electrodes may range from about 10 nm toabout 1 um; in some embodiments, the electrodes are about 10 nm to about1 mm; in other embodiments, they are about 20 nm to about 100 um; inother embodiments, they are about 25 nm to about 1 um thick. Within amembrane, the electrodes may be fabricated of the same or differentmaterials, and they may be the same size or different sizes.

Other means for generating a useful electrostatic field include but arenot limited to grafting polymers, electret deposition and polarization,attachment of proteins and polymers which are negatively charged atphysiologic pH (approximately 7.00-7.50).

3. Pumps

Fabrication of the pores by well known MEMS techniques lends itself tothe integration of such a membrane with previously realized pumps,electrokinetic pumps, pressure sensors, valves, etc. Thus, in someembodiments, the present invention also provides a system as describedbelow, where the membrane and/or system further comprises microscopicperistaltic pumps, configured to direct the movement and flow of fluids.The pumps are generated by nanofabrication with “soft lithography,”using techniques known in the art.

4. Actuators

The use of silicon micromachining techniques lends itself to theaddition of devices to monitor or clean the membrane by thermal,acoustic, electrical or mechanical means. Thus, in some embodiments, thepresent invention also provides a system as described below, where themembrane and/or system further comprises actuators.

In the system of the present invention as described above, the nanoscaleactuators and electronic elements incorporated during nanofabricationare utilized together to limit or reverse protein fouling of the pore,permitting prolonged or indefinite service lifetimes for a filtrationdevice.

5. Monitoring and Control Systems

The systems and devices of the present invention may employ hardwareand/or software monitoring and control systems that collect data,analyze, and report on one or more operations of the system or deviceand/or one or more physiological characteristics of the subject beingtreated. In some embodiments, the monitoring and control systemregulates the operation of the devices/system. The control system maycomprise any combination of software or hardware to carry out itsfunctions. These may be included in the device or as part of a separatedevice.

For example, in some embodiments, the control component is used inconjunction with implanted devices to control fluid conduits havingactively regulated conduit diameters for real-time control of resistanceto fluid flow and hydrostatic pressure in the hemofilter and bioreactor.Integrated fault sensing and safety controls may be used to isolate thedevice from the vasculature in the event of a membrane failure or otherproblem.

The control component may also be used to control/monitor extracellularfluid volume. Control of extracellular fluid volume can be broken downinto three tasks: ECF sensing, selection of feedback algorithm, andeffector mechanism. In hemodialysis, ECF volume is sensed by weighingthe patient and by physical examination. In an implantable device, thisinformation and be used or supplemented with hemodynamic monitoring,including arterial and venous pressure waveform analysis andbioimpedance measurements. For example, a patient may enter his or herweight through a patient data interface, and a computerized algorithmprograms the device to divert a volume of ultrafiltrate from thebioreactor directly to a urinary collection system, be it a bladderanastamosis or a collection pouch. In some embodiments, as thebioartificial kidney may have direct connections to the arterial andvenous vasculature, real-time arterial and venous pressure waveformmonitoring is used, with programmed ultrafiltrate diversion to controlECF volume. A patient interface may use principles commonly used withpacemakers and AICDs (automatic implantable cardio-defibrillators).

II. Systems

The present invention also provides a system comprising a compartmentand the porous membrane of the present invention as described above(e.g., a nanoporous membrane having slit pores and a PEG surfacecoating), where the porous membrane is supported within the compartmentof a device, such that the presence of the membrane separates thecompartment into two sub-compartments. In some embodiments, the systemis a device with a housing, where the housing defines the outer surfacesof the compartments. A housing may be composed of any desired material.Where the system is used on or in a subject, the housing is preferablymade of or coated with a biocompatible material.

The compartment is of any appropriate shape and configuration such thatthe membrane within the device compartment forms two sub-compartmentsthat are completely separate from each other, except that a firstsub-compartment is in fluid connection with a second sub-compartmentonly by means of the pores within the membrane. In preferredembodiments, the device further comprises means for permitting entryinto the first sub-compartment of a first fluid to be filtered (e.g., afeed fluid), and a means for permitting exit of excess feed fluid afterfiltration or of retentate, where the retentate did not get filteredthrough the membrane. In some embodiments, the device further comprisesmeans for permitting exit of a second fluid from the secondsub-compartment, where the second fluid is an “ultrafiltrate” or“permeate” generated from the feed fluid by means of the pores of themembrane, and optionally means for entry into the second sub-compartmentof a third fluid, where such third fluid is a dialyzing fluid for thefeed fluid.

Means for permitting entry of fluid into the first and secondsub-compartments include but are not limited to an opening in thehousing, on one side of the membrane; if such means in bothsub-compartments comprise an opening, then one opening is in either sideof the membrane. The opening may be of any suitable configuration,including but not limiting spheroid, elliptical, and slit-like. Meansfor permitting exit of fluid from the first and the secondsub-compartments include but are not limited to the means for permittingentry of fluid as described above. The entry and exit means are suitablypositioned in the housing to allow entry of fluid, filtration, and exitof fluid, from either or both sub-compartments. The entry and exit meansmay further comprise conduits for delivering fluid to thesub-compartments; such conduits include but are not limited to tubing.When present, such tubing may be inserted into the entry and or exitmeans, or they may be attached to the entry and/or exit means in anyfashion, such as by a clamp or threaded connection, which forms afluid-tight seal of the tubing with the entry and/or exit means.

In further embodiments, the membrane of the device of the invention asdescribed above further comprises at least one surface treatment, asdescribed above. In some embodiments, the surface treatment comprisesattaching cells to the surface of the membrane, as described above. Inthese embodiments, it is contemplated the membrane is used as ascaffolding for cells to process the permeate, for example as isdescribed in U.S. Pat. Nos. 5,549,674, 5,686,289, 6,060,270, 6,150,164,and 6,410,320, the disclosures of which are incorporated herein byreference in their entireties.

In other further embodiments, the membrane of the device of theinvention as described above further comprises means for generating anelectrostatic field, as described above. If desired, the device mayfurther comprise electronic components, for example, amplifiers,filters, transmitters and/or signal preconditioning components. In someembodiments, such components can be incorporated onto the surface of themembrane. In particular, if the membrane comprises elemental silicon,well known integrated circuit technology may be used to place all thecircuitry in miniaturized form on a single chip, which is incorporatedinto the membrane or placed onto and/or attached to the surface of themembrane.

In yet other further embodiments, the membrane of the device of theinvention as described above further comprises at least one surfacetreatment (e.g., with PEG), and at least one means for generating anelectrostatic field.

III. Uses

The membranes of the present invention (e.g., a nanoporous membranehaving slit pores and a PEG surface coating) are able to filter anyfluid from which it is desired to filter one or more types of molecules.The size, shape, array pattern, and charge across a pore are selected inaccordance with the molecules to be filtered. Fluids that can befiltered include but are not limited to biological fluids, includingblood and plasma. Illustrative, non-limiting uses are described below tohighlight the flexibility of the present invention.

A. Hemofiltration

The kidney's functional unit, the nephron, provides for elimination ofwastes and toxins without the need for specific enzymes and transportersfor each toxin. All but the large proteins and cellular elements in theblood are filtered; a system of cells then reclaims specific filteredsubstances needed by the body, and allows all others to pass as urine.Filtration is accomplished by the glomerulus, a tuft of capillariessupported by a basement membrane and specialized epithelial cells calledpodocytes. The filtrate is then passed to the renal proximal tubule, ahollow tube of cells surrounded by capillaries, which accomplishes thebulk of reclamation, as well as other metabolic functions, includingexcretion of acid as various products.

In preferred embodiments, the ultrafiltration devices of the presentinvention having nanoporous membranes (e.g., a nanoporous membranehaving slit pores and a PEG surface coating) are used forhemofiltration. In preferred embodiments, such ultrafiltration deviceshaving, for example, nanoporous membranes with slit pores and a PEGsurface coating reproduce the filtration functions of the native kidney.

In operation, blood is directed from a patient's vasculature, in eitheran extra- or intra-corporeal circuit, into the first sub-compartment ofthe device. After the blood is filtered, it exits the firstsub-compartment, and is returned to/is directed back into patient'svasculature. The route of the blood from the patient through the deviceand back into the patient is referred to as the “blood flow.” In some ofthese embodiments, the blood flow may be assisted or directed by pumps.In some of these embodiments, an ultrafiltrate free of proteins isformed by hydrostatic pressure of blood against the membrane. In some ofthese embodiments, the ultrafiltrate fills the second sub-compartmentduring filtration, and then exits the sub-compartment. In someembodiments, the exit means for the ultrafiltrate include but are notlimited to extraction and draining, where draining may be either byactive or passive means. In yet additional embodiments, theultrafiltrate may be channeled to further devices, which include but arenot limited to testing devices and bioreactors, or it may be removed fordisposal. Removal may be either intracorporeally, as for example bydiversion to the bladder, ileal pouch, or other anatomic conduit, orextracorporeally, as to an external pouch.

In some of these embodiments, the membrane pores, and/or either or bothsurfaces of the membrane itself, are kept free of debris byelectrostatic or electromechanical devices as described above. Themembrane is kept free of debris either by preventing the debris fromaccumulating on the surface, as for example via the PEG surface coating,and/or by maintenance of a steady electrical current, or by removingaccumulated debris, as for example by administering intermittentelectrical current or pulses of current, which may also, in turn, excitean actuator.

By means of the device of the present invention as described above, thedevice mimics the native filtration function of the kidney by producingan ultrafiltrate of plasma similar to that produced by a kidney.Moreover, the ability to prevent fouling of the membrane (e.g., via thePEG membrane surface coating) results in a long service life from themembrane, such that the membrane can be incorporated within a permanentimplantable artificial kidney.

Other filtration applications to which it may be suited are alsocontemplated.

B. Diagnostic Uses

The ultrafiltration devices of the present invention also find use indiagnostic applications. For example, the devices provides a means forselectively screening out undesired molecules (e.g., proteins) withinfluids, such that a particular analyte to be analyzed (e.g., smallmolecules such as glucose, electrolytes, ions, etc.) in the absence ofinterfering molecules. For example, present electrochemical sensors forglucose measurement are severely hampered by protein fouling of thesensor, and great effort is devoted to the invention of foulingretardants to prolong sensor life. An ultrafiltrate substantially freeof proteins, but still containing smaller constituents of blood,including but not limited to sodium, potassium, chloride, glucose,provides a solution to assay for glucose concentration without proteinfouling.

The device may be used to detect any desired analyte. In someembodiments, the analyte is a small molecule. In other embodiments, theanalyte is a pathogen or a molecule or molecular complex associated withthe presence of a pathogen in a sample (e.g., in a blood sample).

In some embodiments, the diagnostic devices are applied on or in asubject for monitoring the presence of or amount of an analyte ofinterest. For example, a glucose or electrolyte sensor monitors (e.g.,at one or more time points or continuously) blood analyte levels. Aprocessor associated with the device reports this information to thesubject or to the appropriate medical personnel (e.g., by displaying theanalyte concentration or by transmitting the analyte concentration—e.g.,to a computer, PDA, phone, or other device). In some embodiments, theprocessor triggers, where appropriate, release of a drug or othersubstance (e.g., insulin) based on the measured concentration so as toalter the physiology of the subject appropriately. In some embodiments,changes in analyte concentration are measured in response to changes inthe environment (e.g., ambient environment, diet, etc) or uponadministration of test compounds (e.g., drugs) to the subject (e.g., fortesting the safety or efficacy of drugs).

In other preferred embodiments, the device is associated with anothermedical device (e.g., a catheter) that is used for in vitro or in vivodetection of the desired analyte. The sensors of the present invention,provide over existing sensor technology (e.g., U.S. Pat. No. 6,405,066,herein incorporated by reference in its entirety).

C. Bioreactors

In some embodiments, the system is used as a convectively fed bioreactorfor cell growth and tissue engineering, for example, as described inU.S. patent application Ser. No. 09/949,575 (Humes et al.), the contentsof which are herein incorporated by reference in their entirety. In somesuch embodiments, cells or tissues are applied to a surface (e.g., amembrane, a chamber surface) or are maintained in suspension in achamber, such that one or more desired fluid flows from the system areexposed to the cells (e.g., exposure of filtered or unfilteredbiological fluids to the cells). In some embodiments, the system isconfigured to permit the exposure of synthetic growth media (e.g., withor without serum) to the cells, alone, or in combination with filteredor unfiltered biological fluid. In some embodiments, the cells aretransgenic cells. In some embodiments, the system is used as a screeningsystem to select cells, genes, drugs, proteins, and/or growth conditionswith desired characteristics and properties.

The cells or tissues may also be used to express or provide one or moredesired factors to a filtered biological fluid that is to be returned toa subject or otherwise manipulated or analyzed.

IV. Bioartificial Organs

The present invention also provides bioartificial organs for in vivo orextracorporeal uses. In some embodiments, the bioartificial organscomprise cells attached to or associated with a surface of a device. Insome such embodiments, the surface is modified (e.g., modified with PEG)to control the biological activity of the attached or associated cells.In some preferred embodiments, the surface is a membrane of the presentinvention, having slit pores, as described herein (e.g., a nanoporousmembrane having slit pores and a PEG surface coating). However, thepresent invention is not limited to the use of surfaces that comprisethe membranes of the present invention. In preferred embodiments, thedevices are configured to combine hemofiltration with cell therapy in amanner that mimics or supplements the function of a healthy organ.

In some embodiments, the cells of the bioartificial organ are suppliedwith nutrients by an ultrafiltrate stream generated by ultrafiltrationof blood or body fluids by a membrane of the present invention. In otherembodiments the cells and tissues of the bioartificial organ are grownon or attached to a membrane of the present invention. In otherembodiments the cells and tissues of the bioartificial organ are grownon or attached to a membrane of the present invention and the cells ofthe bioartificial organ are supplied with nutrients by an ultrafiltratestream generated by ultrafiltration of blood or body fluids by a secondmembrane of the present invention.

In preferred embodiments, the bioartificial organ is a bioartificialkidney. Such devices, find use, for example, in the treatment ofend-stage renal disease. The compact nature of the devices of thepresent invention allows for in vivo or easy, portable, extracorporealtreatment. In-center dialysis, the most common mode of treatment ofend-stage renal disease, is expensive and labor-intensive. Thus, theminiature devices of the present invention simplify, improve, orrelocate to home or in vivo, the treatment of end-stage renal disease,resulting in cost savings and improved quality of life for treatedsubjects. Thus, the present invention provides advantages over orextensions to existing bioartificial kidneys (see e.g., U.S. Pat. No.6,150,164, herein incorporated by reference in its entirety).

A. Surfaces

In some embodiments, the devices comprise a surface for the growth ofcells (see e.g., section III, C above describing bioreactors). Thepresent invention is not limited by the nature of the surface on whichthe cells are grown. Any surface that permits cell to have desiredbiological properties (e.g., attachment, growth, cell division, proteinproduction, protein secretion, membrane fluidity, endocytosis, etc.) iscontemplated by the present invention. The material properties uponwhich cells are grown influence cell attachment and differentiation.This includes geometric patterning and distribution of ECM bindingproteins, surface topology, and porosity of the surface. In someembodiments, the surfaces are coated with self-assembling monolayers,multilayers, or particles. A wide variety of patterned self-assemblingmaterials are known (see e.g., Mrksich, Chem. Soc. Rev., 29:267 (2000)and U.S. Pat. No. 6,017,390). The coating used on the surfaces cancomprise or provide and attachment site for ligands for selectiveprotein/cell attachment or rejection, or otherwise selectively attractor reject desired or undesired molecules or materials.

Examples of surface modification that allow one to tailor the propertiesof the associated cells are found in Desai, Med. Eng. Phys. 22:595,2000, Deutsch et al., J. Biomed. Mater. Res., 53:267, 2000, Kapur etal., J. Biomed. Mater. Res., 33:205, 1996, Brunette and Chehroudi,121:49, 1999, Brunette, Exp. Cell Res., 167:203, 1986, Brunette, Exp.Cell Res. 164:11, 1986, den Braber et al., J. Biomed. Mater. Res.,29:511, 1995, den Braber et al., J. Biomed. Mater. Res., 17:2037, 1996,Curtis and Wilkinson, Biomaterials 18:1573, 1998, Craighead et al.,Biomed. Microdevices, 1:49, 1998, Mata et al., Biomed. Microdevices4:267, 2002, Mata et al., J. Biomed. Mater. Res., 62:499, 2002, and U.S.Pat. Nos. 5,776,748, 5,843,741, 5,976,826, 6,569,654, 5,770,193,5,759,830, 5,736,372, and 5,770,417, each of which is hereinincorporated by reference in their entireties.

In some preferred embodiments, the surface is a membrane of the presentinvention (see e.g., section I, above) (e.g., a nanoporous membranehaving slit pores and a PEG surface coating). Use of such membranesprovides a number of advantages, including the ability to miniaturizethe bioartificial device to allow in vivo use or efficient andconvenient extracorporeal use. An example of a nanoporous membrane foruse in the bioartificial organs of the present invention is described inthe Examples.

The surface of may be precoated with suitable extracellular matrix (ECM)components including Type I collagen, Type IV collagen, laminin,Matrigel, proteoglycan (such as heparin sulfate and dermatan sulfate)fibronectin, and combinations thereof to form an ECM layer. Once an ECMlayer has been established on the surface, this layer is then seededwith desired cells.

B. Cells

A variety of cells find use in the bioartificial organs of the presentinvention. In some embodiments the cells of the bioartificial organ areliver, duodenal, intestinal, gastric, pancreatic, thyroid, parathyroid,adrenal, gonadal, pituitary, or hypothalamic cells. In some embodimentsthe cells of the artificial organ are bone marrow cells. In otherembodiments the cells of the bioartificial organ are stem cells, feedercells, or other precursor cells. In still other embodiments, the cellsof the bioartificial organ are derived from stem or precursor cells. Instill other embodiments, the bioartificial organ comprises cells thatinduce the differentiation of nearby cells or attract nearby cells tothe organ. In some embodiments, the cells comprise one or moretransgenes (e.g., having inducible promoters).

In preferred embodiments, the cells are from kidney or associatedtissue. Cells from many segments of the nephron have been grown inprimary culture (see for example, Handler & Burg in “Application oftissue culture techniques to study of renal tubular epithelia” inWindhager & Giebisch (eds):Handbook of Physiology, Section 8, RenalPhysiology, American Physiological Society, Williams & Wilkins,Baltimore; incorporated herein by reference in its entirety). Specificcells have been separated on the basis of differential growth, bymechanical dissection, by differential centrifugation and with the aidof specific antibodies (immunodissection).

In some preferred embodiments, the cells are renal proximal tubulecells. These cells replace the metabolic, endocrine, and immunologicfunctions of a damaged kidney. Cells are grown on the appropriatesurface and then exposed to ultrafiltrate. The cell-exposedultrafiltrate is then returned to a subject. It is contemplated that thecell-exposed ultrafiltrate contains serum appropriate levels of desiredbiological components (e.g., 1,25 dihydroxy-vitamin D₃, sodium, glucose,etc.).

In some embodiments, a mixture of cell types is associated with thesurface. In some such embodiments, a first layer of a first cell type isgrown, which provides a new surface for the growth a second oradditional cell types. For example, pericyte, vascular smooth muscle ormesangial cells can be first seeded on a ECM layer and allowed to reachconfluence. Thereafter, endothelial or other cells can be seeded.Pericyte cells are described by Sims in Can. J. Cardiol. 7(10):431-443(1991) and Shepro et al in FASEB J. 7:1031-1038 (1993), incorporatedherein by reference. Mesangial cells, the preferred type of pericytecell, are described by Davies in Kidney International, 45:320-327(1994), incorporated herein by reference.

Suitable culturing techniques useful for seeding these cells on thesurface are described by Scott et al., J. Cell Sci. 105:269-273, 1993;Schneider et al., Surgery 103:456-462, 1988; Kadletz et al., J. Thoracicand Cardiovascular Surgery 104:736-742, 1 1992; Shepard et al., Surgery99: 318-3.about.6, 1986; and Demetriou et al., Science 23:1190-1192,1986; each of which is incorporated herein by reference in theirentireties.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Nanofabrication of Membranes

This example describes the process flow for fabrication of nanoporousmembranes; this process is depicted in FIG. 1. The starting substrate isa 400 μm-thick, 100 mm-diameter, double side polished (100)-orientedsilicon wafer that is obtained from a commercial vendor of semiconductorsubstrates. The wafer is coated with a 5000 Å-thick layer of low-stresssilicon nitride (LSN) by low-pressure chemical vapor deposition (LPCVD).Next, a 4 μm-thick film of polysilicon is deposited by LPCVD (FIG. 1(a))and followed by thermal oxidation to grow a 2500 Å-thick layer of SiO₂.The oxide layer on the wafer front side is then patterned byphotolithography and wet etching in buffered hydrofluoric acid (BHF) tocreate an etch mask, which is used to pattern the underlying polysiliconfilm by reactive ion etching (RIE) in chlorine plasma. Afterwards, BHFis used to remove the masking oxide on both wafer front and back sidesand followed by RIE to remove polysilicon on the wafer back side (FIG.1(b)). Next, thermal oxidation is performed to realize a 20 nm-thickSiO₂ film that will define the pore size in the nanomembrane (FIG.1(c)). It should be noted that other pore sizes, if desired, could berealized by varying the thickness of the SiO₂. The anchor regions arethen defined by selectively patterning the oxide on the wafer frontsideusing photolithography and BHF. Next, another 4 mm-thick polysiliconfilm is deposited by LPCVD (FIG. 1(d)) and followed by globalplanarization by chemical-mechanical polishing (CMP) to remove anyexcess polysilicon and expose the pore regions on the frontside (FIG.1(e)). The polysilicon and LSN on the backside are then removed by RIEin chlorine and SF6 plasma, respectively, and followed by a LPCVDdeposition of LSN on both front and back sides of the wafer (FIG. 1(f)).Afterwards, the LSN on the wafer backside is patterned usingphotolithography and RIE to define an etch mask (FIG. 1(g)) for thesubsequent KOH etch to create suspended membranes (FIG. 1(h)). Finally,the masking LSN and SiO₂ films are etched in concentrated hydrofluoricacid to realize the nanoporous membranes (FIG. 1(i)).

In additional experiments, the design and construction of polysiliconmembranes with 10 to 100 nm pores was characterized. The nanoporousmembranes were fabricated by standard silicon bulk and surfacemicromachining processes. The pore structure was defined by depositionand patterning of a polysilicon film on the silicon wafer. The criticalsubmicron pore dimension is defined by the thickness of a sacrificialSiO₂ layer, which was grown with unprecedented control to within +/−1nm. The oxide layer was etched away in the final processing step tocreate the porous polysilicon nanomembrane. Membranes were mounted onpolycarbonate filter inserts and examined under light microscopy forbreaks or pinholes. Carriers were inserted into an Ussing chamber devicefitted with pressure transducers, and both sides of the membrane wereprimed with aqueous solution. One side of the chamber was connected to acollection vessel at atmospheric pressure, and the other to a calibratedsyringe. Syringe pumps were used to deliver fluid at set rates to themembrane, and the pressure generated by flux through the membrane wasmeasured. Agreement was obtained between the observed and predictedhydraulic resistance. The hydraulic permeability was similar to that ofcommercial ultrafiltration membranes, suggesting that repeatablepressure-driven hydraulic flows may be observed in micro- andnano-machined membranes (FIG. 2).

Example 2 Extracorporeal Hemofiltration

This example demonstrates how a nanofabricated nanoporous membrane maybe used to form an extracorporeal hemofiltration device (see e.g., FIG.3). Blood from a patient or from a stored supply is directed to anorifice 600 by means of a cannula, catheter or other means. An optionalpump 100, which may be peristaltic, rotary, roller, or other, is used toregulate a flow of blood to a chamber 700, which contains a pressuresensor 401 and is bounded by a membrane 201 composed of a plurality ofpores. Said pores may be shaped to optimize hydraulic permeability, andmay be all alike or dissimilar. Furthermore, said pores may contain orcomprise electrodes, surface treatments, or be coated with chemicals,polymers, proteins, sugars, and the like to impart a particularelectrostatic charge to the pore or a region around the pore, and impartan electric field within the pore. Blood exits the chamber 701 via anorifice 602 with an optional pump 102 which may be peristaltic, rotary,roller, or other, and is returned to the patient or to a reservoir viacannula, catheter or other means. Fluids, in this example an electrolytesolution, or optionally an anticoagulant solution, or other solution notspecified may be introduced into the blood in chamber 701 via orifice orinlet 602 and optional pump or valve 101. The pressure sensor 401, incombination with external or integrated electronics and controls, withvalves and pumps 100, 101, and 102 may be used to regulate flow of bloodinto and out of chamber 701, and specifically to regulate and adjust thehydrostatic pressure in chamber 701. A second chamber 702 is positionedto receive filtrate passing through the membrane 201 either under forceof hydrostatic pressure or eletroosmotic flow or other means notspecified. Chamber 702 incorporates a second pressure sensor 402, asensor or array of sensors 302 incorporating but not limited to optical,conductance, impedance, magnetic resonance, electrochemical, orimmunologic principles, and a conduit 603 and pump or valve 103 forremoval of fluid to a reservoir or drain. The sensor or array of sensors302 may be used to monitor the composition of the ultrafiltrate andactuate alarms, valves, or other devices, including but not limited totelemetry and telephony devices, in event that a parameter measured ofthe ultrafiltrate falls out of a prescribed range. In this fashion ananofabricated nanoporous membrane may be used to accomplishhemofiltration of blood.

Example 3 Continuous Blood Glucose Sensor

This example demonstrates how a membrane may be used to form acontinuous blood glucose sensor. The novelty and advantage of thisapproach is the rapidity with which the glucose level in the blood istransmitted to the sensor, as glucose is carried by convection to thesensor, rather than by diffusion towards the sensor, while stillaffording the sensor protection from elements in the blood that may beinjurious to or degrade the sensor. The example of a blood glucosesensor is not to be construed as limiting the application; it may beapplied to the analysis of cell and/or protein free fluids for arbitraryanalytes by arbitrary means. A preferred embodiment is illustrated inFIG. 4. Blood from the patient is directed by means of a cannula, avascular anastamosis, a synthetic graft, or other means to an inlet 600optionally equipped with a pump or valve or other flow controller 100 toa chamber 700, which optionally contains a pressure sensor 401 and isbounded by a membrane 201 composed of a plurality of pores. Said poresmay be shaped to optimize hydraulic permeability, and may be all alikeor dissimilar. Furthermore, said pores may contain or compriseelectrodes, surface treatments, or be coated with chemicals, polymers,proteins, sugars to impart a particular electrostatic charge to the poreor a region around the pore, and impart an electric field within thepore. A second cannula or vascular anastamosis, or synthetic graft orother means returns blood from the chamber via an optional flowcontrolling device 102 and outlet 602 to the patient's blood stream. Thepressure sensor 401, in combination external or integrated electronicsand controls, with valves and pumps 100 and 102 may be used to regulateflow of blood into and out of chamber 700, and specifically to regulateand adjust the hydrostatic pressure in chamber 700. A second chamber 702is positioned to receive filtrate passing through the membrane 201, andoptionally incorporates a second pressure sensor 401, and a sensor orarray of sensors 302 incorporating but not limited to optical,conductance, impedance, magnetic resonance, electrochemical, orimmunologic principles. In the present Example, at least one of thesensors 302 is able to measure the concentration of glucose in theultrafiltrate. The ultrafiltrate then exits the second chamber, eitherunder hydrostatic pressure or by means of an active pump or valve 103and is directed to an outlet 603 which joins with and is continuous withoutlet 602 returning blood from the first chamber 700 to the patient'sblood stream by means of a cannula or vascular anastamosis, or syntheticgraft or other means. The sensor or array of sensors 302 may be used tomonitor the composition of the ultrafiltrate and actuate alarms, valves,or other devices, including but not limited to telemetry and telephonydevices, in event that a parameter measured of the ultrafiltrate fallsout of a prescribed range. In the present example, the sensor would beconnected to central processing unit incorporating a digital-to-analogconverter and a means, such as an antenna or a light emitting device(LED) for transmitting the value measured by the sensor through thepatients skin by electromagnetic or optical means, for detection,recording, and analysis by the patient or others. In this way, theinvention may be used to construct an indwelling blood glucose sensorcapable of continuous measurement of glucose levels, although theprinciple is general and it may be easily seen to extend to themeasurement of any analyte of size and charge such that it may be passedthrough a membrane designed for such purpose.

Example 4 Bioartificial Kidney

This example demonstrates how nanofabricated nanoporous membranes may beused to form a bioartifical kidney device. A preferred embodiment isshown in FIG. 5. Two membranes 201 and 202 are housed in a housing 10.Blood or other body fluid from a patient is directed via a cannula,vascular graft, vascular anastamosis, or other method into an orifice600 containing an optional pump or valve 100, which may be peristaltic,rotary, roller, or other, and may be used to regulate a flow of fluid toa chamber in the housing 701, which contains a pressure sensor 401; amembrane 201 composed of a plurality of pores; and an outlet 601 with aflow controlling device such as a pump or valve 101. Said pores may beshaped to optimize hydraulic permeability, and may be all alike ordissimilar. Furthermore, said pores may contain or comprise electrodes,surface treatments, or be coated with chemicals, polymers, proteins,sugars to impart a particular electrostatic charge to the pore or aregion around the pore, and impart an electric field within the pore.The outlet 601 and flow controller 101 may be used in conjunction withpressure sensor 401 and pump, valve, or flow controller 100, andexternal or integrated electronics, telemetry, and informationprocessing to regulate flow of blood or body fluids into and out ofchamber 701, and in particular to regulate the hydrostatic pressure inchamber 701. The outlet 601 and flow controller 101 control flow ofblood into a second chamber 702, which is equipped with a pressuresensor 402; optionally other sensors incorporating but not limited tooptical, conductance, impedance, magnetic resonance, electrochemical, orimmunologic principles; and an outlet 602 containing a flow regulatingdevice such as a pump or valve 102. Outlet 602 and its associated flowcontroller 102 may be used in conjunction with pressure sensor 402 andother pressure sensors and flow controllers and external or integratedelectronics, telemetry, and information processing to regulate flow ofblood or body fluids into and out of chamber 702, and in particular toregulate the hydrostatic pressure in chamber 702. Blood or body fluidsexiting orifice 602 is returned to the patient via a cannula, vasculargraft, vascular anastamosis, or other method.

A third chamber 703 is positioned to receive ultrafiltrate generated byhydrostatic pressure or electrosmotic flow of blood or body fluid inchamber 701 passing through the membrane 201, and incorporates a secondpressure sensor 403; a sensor or array of sensors 303 incorporating butnot limited to optical, conductance, impedance, magnetic resonance,electrochemical, or immunologic principles; and an outlet 603 and flowcontroller 103. In the example of a bioartificial kidney, it iscontemplated that this ultrafiltrate is substantially free of proteinsand cellular elements. Flow controller 103 directs ultrafiltrate to afourth chamber 704, similarly equipped with a pressure sensor 404 andother sensors 304 incorporating but not limited to optical, conductance,impedance, magnetic resonance, electrochemical, or immunologicprinciples, and an outlet 604 with a flow control mechanism 104. Thesensor or array of sensors 304 may be used to monitor the composition ofthe ultrafiltrate and actuate alarms, valves, or other devices,including but not limited to telemetry and telephony devices, in eventthat a parameter measured of the ultrafiltrate falls out of a prescribedrange.

Chambers 702 and 704 are connected by a second membrane 202 which may betreated, coated, adsorbed, or otherwise modified with cells or tissues.For example, in some embodiments, the cells comprise epithelial,endothelial, fibroblast, or other cells. In some embodiments, the cellsare transgenic cells that are engineered to express or not expressdesired genes (e.g., to modulate the secretion of proteins or othersecreted molecules, to express extracellular molecules that bind desiredligands, etc.). In some embodiments, the membrane 202 is also associatedwith sorbents, enzymes, proteins, channels, porins, or other agents tocontrol and direct the flow of fluids, electrolytes, toxins, peptides,proteins, or other chemicals, through said membrane 202 and into chamber702 where such fluids, electrolytes, toxins, peptides, proteins, orother chemicals mix with the blood or body fluid that has enteredchamber 702 via orifice 601. Blood or body fluid that has been mixedwith the cellular and metabolic products of the membrane 202 is thenreturned to the patient via orifice 602 as described. The ultrafiltratewhich has been processed by the second membrane but has not beenreabsorbed is carried away from chamber 704 via an outlet 604 and isthen carried to a reservoir or to the patient's urinary bladder, anenteric loop, or other suitable disposal route. Through this means, aswell as others not specified herein, a patients bloodstream may befiltered and processed to remove solutes, toxins, electrolytes, andwater while preserving circulating volume, small peptides, amino acids,and other molecules essential to homeostasis.

A sacrificial oxide technique was used to fabricate arrays of 1 mm×1 mmsilicon membranes with 10-100 nm×45 μm slit pores. There wereapproximately 10⁴ slit pores per array. After etching away thesacrificial oxide, the membranes were epoxied to an acrylic orpolycarbonate carrier and inspected via light microscopy for defects. Acustom-built apparatus was used to test the membranes. Acrylic wasmachined to provide two cylindrical half-chambers, each with inlet andoutlet Luer fittings. A pressure transducer (Omega PX61) was threadedinto a separate port in one chamber. The two halves were boltedtogether, trapping the membrane and carrier between. Buna-N O-ringsprovided watertight and gastight seals between the two half-chambers andthe membrane carrier. A Luer manifold system allowed regulation of fluidflow into each half chamber. Driving force for gas flow was provided bycompressed gas cylinders and for liquid flow by a peristaltic pump.Independent control of flow rate into each chamber and pressure withineach chamber was achieved by varying the diameter of tubing draining thechamber. The volumetric flows of gases and liquid were measured bytiming positive displacement of a liquid meniscus in calibrated pipettesor syringes.

Nitrogen and carbon dioxide were individually used to flush both sidesof the membranes. The outlet of the feed side and the inlet of thepermeate side were closed. The outlet on the permeate side was connectedto the top of a pipette filled with vacuum oil. The feed side waspressurized at 1.00, 1.25, 1.50, 1.75, and 2.00 psi, and the downwarddisplacement of oil was timed at each pressure. By regulating the heightof the meniscus from run to run, the outlet pressure was held to within2-3 cm oil from experiment to experiment. Tests with dummy membraneswithout pores and open membranes with macroscopic holes were alsoconducted to validate the system. The gas flow through the membranes wasused initially to confirm that the membrane pores were open and wasconsistent in performance between and within wafers. Furthermore, carbondioxide is an ideal wetting agent prior to aqueous experiments, as CO₂bubbles readily dissolve into aqueous solution and allow avoidance ofsurface tension issues with nitrogen bubbles. Phosphate buffered saline(PBS) was stored in a reservoir and circulated with a peristaltic pump.After membrane flushing with carbon dioxide to exclude air bubbleswithin the pores, both sides of the membranes were flushed with PBS, andthe inlet port of the permeate side sealed. The outlet port wasconnected to a calibrated syringe barrel, and an oil seal was placed onthe syringe barrel. Flow through the feed side of the chamber wasadjusted to produce transmembrane pressures of 1.00, 1.25, 1.50, 1.75and 2.00 psi. Volumetric displacement of the PBS-air meniscus under theoil seal was timed to calculate volume flow. Pressure-flow curves weregenerated for each pore size and hydraulic permeabilities for PBS werecalculated. Measured hydraulic permeabilities correlated well withNavier-Stokes predictions for Hele-Shaw flows (Fissell et al., J. Amer.Soc. Nephrology, vol. 13, pp. 602A, 2002). Also noteworthy were thesimilarities in hydraulic permeabilities (Kuf) of the silicon nanoporousmembranes and commercial polymer dialysis membranes (Fresenius andBaxter). This is particularly interesting considering that the siliconmembranes have a porosity that is orders of magnitude smaller than thatof polymer membranes.

Silicon chips 1×1 cm square were diced from a 100 mm diameter, 500 μmthick, <100>-oriented n-type single-side polished wafer. Similarly, 1×1cm square chips of polycrystalline silicon (polysilicon) were diced froma 100 mm diameter, <100>-oriented, n-type single side polished waferthat was oxidized to grow a 1000 Å-thick oxide layer followed by thedeposition of a 5 μm thick polysilicon film by low-pressure chemicalvapor deposition. Murine collagen IV and fetal calf serum werenonspecifically adsorbed onto steam-autoclaved silicon and polysiliconchips, which were placed in 12 mm-diameter tissue culture wells. Humanrenal proximal tubule cells (RPTCs) were harvested from transplantdiscards and grown to fourth passage on 100 mm-diameter tissue cultureplates, resuspended, and stained with a fluorescent cell linker(PKH26-GL, Sigma, St. Louis) (Humes et al., Amer. J. Physiology,271:F42, 1996). Aliquots of 10⁵ cells were layered onto silicon andpolysilicon chips with preadsorbed extracellular matrix proteins.

Cell growth was monitored by light microscopy in control wells. Whencells reached approximately 75% confluence, 90% confluence, and completeconfluence, chips were removed from tissue culture media and fixed incold 4% paraformaldehyde for 20 minutes and then rinsed with coldphosphate buffered saline and stored in PBS at 0° C. Renal proximaltubule cells were observed to attach to single-crystal silicon andpolysilicon chips when pretreated with ECM proteins, and retain surfacemarkers characteristic of renal proximal tubule cells, including tightjunction proteins. Specifically, areas of the silicon chips where themembranes were open and porous (M) were compared with areas where thesilicon surface was identically textured and prepared, but amonocrystalline silicon backing layer occluded the pores (S). Siliconchips bearing membranes upon which HPTCs had been grown to confluencewere incubated with antibodies to two protein markers of differentiation(acetylated tubulin (AT1) and ZO-1). Fluorescently labeled secondaryantibodies were then used to examine the cells by immunofluorescencemicroscopy. A fluorescent marker for cell nuclei (DAPI) was used as acontrol. Cells attached to S areas and M areas in approximately equaldensity, and intensity of fluorescence of the DAPI stain did not varyappreciably between S areas and M areas. ZO-1 expression on the surfacesof HPTCs in M areas was increased compared with S areas, although at thetime of cell fixation it had not localized to intercellular junctions.Intensity of fluorescence of DAPI was similar between the two areas.Acetylated tubulin is a component of the primary cilium of renalproximal tubule cells. Acetylated tubulin staining in M areas was moreintense than in S areas, although DAPI staining remained uniform inintensity over the two areas. These observations show that detailedstructuring of surface textures and porosity of silicon nanoporousmembranes has direct impact of cellular differentiation.

Example 5 Nanoporous Membranes for Bioartifical Organs

This example demonstrates how nanofabricated nanoporous membranes may beused to form a bioartifical kidney device. A preferred embodiment isshown in FIG. 6. Two membranes 201 and 202 are housed in a housing 10.Blood or other body fluid from a patient is directed via a cannula,vascular graft, vascular anastamosis, or other method into an orifice600 containing an optional pump or valve 100, which may be peristaltic,rotary, roller, or other, and may be used to regulate a flow of fluid toa chamber in the housing 701, which contains a pressure sensor 401; amembrane 201 composed of a plurality of pores; and an outlet 601 with aflow controlling device such as a pump or valve 101. Said pores may beshaped to optimize hydraulic permeability, and may be all alike ordissimilar. Furthermore, said pores may contain or comprise electrodes,surface treatments, or be coated with chemicals, polymers, proteins,sugars to impart a particular electrostatic charge to the pore or aregion around the pore, and impart an electric field within the pore.The outlet 601 and flow controller 101 may be used in conjunction withpressure sensor 401 and pump, valve, or flow controller 100, andexternal or integrated electronics, telemetry, and informationprocessing to regulate flow of blood or body fluids into and out ofchamber 701, and in particular to regulate the hydrostatic pressure inchamber 701. The outlet 601 and flow controller 101 control flow ofblood into a second chamber 702, which is equipped with a pressuresensor 402; optionally other sensors incorporating but not limited tooptical, conductance, impedance, magnetic resonance, electrochemical, orimmunologic principles; and an outlet 602 containing a flow regulatingdevice such as a pump or valve 102. Outlet 602 and its associated flowcontroller 102 may be used in conjunction with pressure sensor 402 andother pressure sensors and flow controllers and external or integratedelectronics, telemetry, and information processing to regulate flow ofblood or body fluids into and out of chamber 702, and in particular toregulate the hydrostatic pressure in chamber 702. Blood or body fluidsexiting orifice 602 is returned to the patient via a cannula, vasculargraft, vascular anastamosis, or other method.

A third chamber 703 is positioned to receive ultrafiltrate generated byhydrostatic pressure or electrosmotic flow of blood or body fluid inchamber 701 passing through the membrane 201, and incorporates a secondpressure sensor 403; a sensor or array of sensors 303 incorporating butnot limited to optical, conductance, impedance, magnetic resonance,electrochemical, or immunologic principles; and an outlet 603 and flowcontroller 103. In the example of a bioartificial kidney, it iscontemplated that this ultrafiltrate is substantially free of proteinsand cellular elements. Flow controller 103 directs ultrafiltrate to afourth chamber 704, optionally equipped with a pressure sensor and othersensors not shown incorporating but not limited to optical, conductance,impedance, magnetic resonance, electrochemical, or immunologicprinciples. In some embodiments, chamber 704 is fitted withnanofabricated or other assemblies 204, which may be treated, coated,adsorbed, or otherwise modified with cells or tissues 500. In someembodiments, these cells may be pancreatic islet cells. In someembodiments these may be hepatocytes. In other embodiments these may betransgenically modified cells, prokaryotic or eukaryotic cells, bonemarrow cells, xenotransplanted cells, allografted cells, or stem cellsof embryonic or adult origin of human or other species. These examplesshall not be construed as limiting the type, variety and mixtures ofcells to be employed. In this example, cells 500 are permitted to bebathed by the ultrafiltrate of blood generated by membrane 201 anddelivered to them from chamber 703 via orifice 603. In some embodiments,said ultrafiltrate is free of immunoglobulins, complement components ofblood, chemotherapeutic agents, or other entities in the blood harmfulto cells 500. Said cells 500 may metabolize toxins in the ultrafiltrate,in the example in which they are hepatocytes, or may sense theconcentration of some entity in the ultrafiltrate, such as glucose, andrespond by secreting a hormone or other molecule, such as insulin. Inanother embodiment, cells 500 may be renal cells that secreteerythropoetin in response to oxygen tension in the ultrafiltrate.Chambers 702 and 704 are connected by a second membrane 202 that may betreated, coated, adsorbed, or otherwise modified with cells or tissues.In some embodiments, the membrane 202 is also associated with sorbents,enzymes, proteins, channels, porins, or other agents to control anddirect the flow of fluids, electrolytes, toxins, peptides, proteins, orother chemicals, through said membrane 202 and into chamber 702 wheresuch fluids, electrolytes, toxins, peptides, proteins, or otherchemicals mix with the blood or body fluid that has entered chamber 702via orifice 601. In some embodiments, the porous structure of membrane202 is designed to prevent passage of a specified protein, peptide,sugar, lipid, bacterium, or other entity into chamber 702. Blood or bodyfluid that has been mixed with the cellular and metabolic products ofthe membrane 202 is then returned to the patient via orifice 602 asdescribed. Through this means, as well as others not specified herein, apatient may receive a dose of cells of arbitrary type while such cellsare protected from the immune effectors in the blood, while receivingconvective transport of nutrients and oxygen from the blood, and thebiological products of such cells may re-enter the patient's bloodstreamin a controlled fashion.

Example 6

This example describes the fabrication of nanopore membranes employingcertain embodiments of the invention. Nanopore membranes withmonodisperse pore size distribution have been prototyped from siliconsubstrates by a process based on MEMS technology (see, e.g., Lopez, C.A., et al., Biomaterials, 2006. 27(16): p. 3075; Fissell, W. H., et al.,Blood Purif, 2007. 25(1): p. 12). The process used the growth of a thinsacrificial SiO₂ (oxide) layer to define the critical submicron poresize of the filter. The oxide was etched away in the final step of thefabrication process to leave behind open pores in the form of, forexample, parallel-plate nanochannels. Thermal oxidation of siliconsubstrates provided oxides down to 5 nm in thickness with <1% variationacross the wafer.

The starting substrate was a 400 μm-thick, 100 mm-diameter, double sidepolished<100>-oriented silicon wafer that was obtained from a commercialvendor of semiconductor substrates. The wafer was coated with a 500nm-thick layer of low-stress silicon nitride (LSN) followed by a 5μm-thick film of polycrystalline silicon (polysilicon) (FIG. 10(a)). Thepolysilicon layer was patterned by photolithography and reactive ionetching (RIE) to create 50 μm-long spaces and lines that were 2-3 μmwide (FIG. 10(b)). Thermal oxidation of the patterned polysilicon wasperformed to grow a thin conformal SiO2 film that defined the pore sizein the nanoporous membrane (FIG. 10(c)). The thickness of this conformalSiO2 film can be readily varied between 5-100 nm by adjusting oxidationparameters such as time and temperature. After deposition of another 4μm-thick polysilicon film, chemicalmechanical polishing was performed toexpose the nanopore regions on the frontside (FIG. 10(e)). LSN wasdeposited on both front and back sides of the wafer (FIG. 10(f)). TheLSN on the wafer backside was patterned to define an etch mask (FIG.10(g)). The wafer was subsequently etched in KOH to create suspendedmembranes (FIG. 10(h)). Finally, the masking LSN and SiO₂ films wereetched in concentrated hydrofluoric acid to produce nanoporous membraneswith 40 μm-long slit pores with nanoscale pore width (FIG. 10(i,j,k)).

Membranes with critical pore widths of 5-100 nm have been routinelyfabricated with <1% mean pore size variation across a 100 mm-diameterwafer (FIG. 10(l)). For experiments, the wafer was sized into 1×1 cmchips bearing arrays of 1×1 mm nanoporous membranes. Each membrane was˜4 μm thick and contained ˜104 slit pores, which translated to overallporosity of ˜1%. Recent advances in nanoimprint lithography technologymake it feasible to generate lines and space patterns down to <50 nm(see, e.g., Guo, L. J., Journal of Physics D: Applied Physics, 2004. 37:p. R123; Tallal, J., et al., Microelectronic Engineering, 2005. 78-79:p. 676). In some embodiments, this capability permits closer porepacking, and increase membrane porosity from ˜1% (2 μm pore separation)to >20% (<100 nm pore separation). In some embodiments, uniform oxidefilms down are grown to 2 nm on singlecrystal silicon (see, e.g.,Bidaud, M., et al., Journal of Non-Crystalline Solids, 2001. 280(1-3):p. 32).

Example 7

This example describes the biocompatibility of MEMS materials. Silicon(Si), silicon dioxide (SiO₂), and silicon carbide (SiC) were examinedusing a battery of standardized in vitro test protocols based on theInternational Standards Organization (ISO) Biocompatibility Guideline(ISO 10933); incorporated herein by reference in its entirety. The MEMElution Test was used to evaluate the materials for potential cytotoxiceffects with black rubber and polypropylene as the positive and negativecontrols, respectively. Extracts of the test materials were added tocell culture plates seeded with L-929 mouse fibroblast or WI-38 humanembryonic lung cells and incubated at 37° C. with 5% CO₂ for 48 hours.Cell culture plates were then examined microscopically (100×) todetermine any change in cell morphology and lysis, monolayer confluency,and color as an indicator of resulting pH. Results were scored on ascale of 0-4, where 0 represented the best case—no adverse reactionwhatsoever—and 4 represented the worst case—complete cell lysis. A scoreof 2 or below was considered acceptable for many implantableapplications.

Negative and positive controls exhibited grades of 0 and 1,respectively. Si, SiO₂, and SiC exhibited a grade of 0 meaning that suchmaterials do not exhibit any cytotoxicity, and could be exposed to thein vivo biological environment. Similar results were obtained for invivo irritation tests from implantation into rabbit musculature.

Si and SiO₂ were subsequently evaluated for hemocompatibility toascertain whether they would adversely induce hemolysis, osmoticfragility, or coagulation. The hemolysis test is a standard method todetermine the amount of blood cell lysis caused by a material sample.Extract samples were incubated with citrated human blood cells fromhealthy donors in 0.9% saline for 1 hour at 37° C. Afterwards intact redblood cells were removed and the optical density of the supernatantfluid was analyzed using a spectrophotometer set at 540 nm. The opticaldensity readings of the sample were normalized to those from thepositive and negative controls to determine net hemolysis due to thematerial being tested. The results showed that Si and SiO₂ did notexhibit hemolysis. The osmotic fragility test is an extension of thestandard hemolysis test and was used to determine the relative increasein hemolysis with decreasing osmotic pressure caused by a materialsample. The results showed that none of the MEMS material samplesinduced osmotic fragility.

The effect of Si and SiO₂ on blood coagulation was examined using thepartial thromboplastin test (PTT) and prothrombin time (PT) test, whichare general screening tests for the detection of abnormalities in theintrinsic and extrinsic pathways, respectively. In the PTT test, thesample extract was incubated with PTT reagent for 3 minutes at 22° C.,and afterwards clotting time was determined after addition of calciumchloride reagent. In the PT test, the extract was added to calciumchloride and thromboplastin reagents and the resulting clotting time isdetermined. Clotting times of the material samples were compared tocorresponding times from polypropylene samples, which act as negativecontrol. The results from the PTT and PT tests showed that SiO₂exhibited a slight variation (p<0.05) in clotting time, while Siexhibited clotting behavior that was consistent with the negativecontrol.

In summary, results of the biocompatibility tests confirm that theprimary constituent materials of the nanoporous membranes—Si and SiO₂;the latter forms on exposed Si surfaces—are suitable for an implantablehemofilter and tubule bioreactor.

Example 8

This example describes surface modification of the nanopore membranes.To explore the functionality of solution phase coupled PEG to thepreviously mentioned nanoporous membranes the method employed by Larsonet al. was utilized (see, e.g., Papra A, et al., Langmuir 17:1457-1460(2001); incorporated herein by reference in its entirety). Thistechnique uses a single-step mechanism which covalently couples siliconsurface silanol groups (Si—OH) to a PEG polymer through atrimethoxysilane group forming a Si—O—Si-PEG sequence by a methanoldehydration reaction. This method is appealing due to the ease ofapplication and availability of commercial materials that can be used asreceived. It is a one step procedure as opposed to other processes suchas CVD, or solution phase coupling that use a bridge molecule to form acovalent bond. For this reason, this deposition technique with slightmodifications was utilized. The modifications included omitting allsonication steps and continuing the PEG deposition for 12 hours.

Resistance of the PEG coatings to protein fouling was examined (takenfrom Sharma et al., 2004 Langmuir 20(2)) with albumin (see, FIG. 8,left) and fibrinogen. Both protein deposition and fibroblast attachment(see, FIG. 8, center) were reduced by 90% on PEG modified substratesrelative to uncoated controls. The optimal grafting surface density wasdetermined to be 20000-25000 PEG molecules per nm² (see, FIG. 8, right).Contact angle measurements confirmed that bare silicon is morehydrophilic (˜10°) compared to PEG modified surfaces (˜350), which isstill less hydrophobic than silanized silicon (˜80°). Also, the contactangle did not depend on the concentration of EtO, suggesting that thesurface is uniformly coated with PEG. Stability of PEG films wasconfirmed over a 4-week period. The modified surfaces (CVD PEG coatings)were kept at 25° C. (room temperature) to study their dry stability andat 37° C. in PBS to study their aqueous stability. The composition ofthe film was determined after 4 weeks. Analysis of the surfaces by x-rayphotoelectron spectroscopy (XPS) indicated that the PEG films remainedextremely stable in the dry environments, while in PBS solution, thecarbon content of the PEG film was reduced by 7%. Contact anglesmeasured after 4 weeks in dry and aqueous conditions was 38±3°,indicating that the PEG modified surface was also stable in terms ofhydrophilicity.

FIG. 9 shows silicon membranes coated with PEG have increased plasmarecalcification time in comparison to silicon membranes lacking PEGcoating and glass slides.

Example 9

This example describes hydraulic permeability testing for the nanoporemembranes. Nanoporous membrane chips (1×1 cm) were epoxied to an acrylicor polycarbonate carrier and mounted in a custom-built apparatus to testthe membranes for fluid transport characteristics (see, FIG. 10).Acrylic was machined to provide two cylindrical half-chambers, each withinlet and outlet Luer fittings. A pressure transducer (Omega PX61) wasthreaded into a separate port in one chamber. The two halves were boltedtogether, trapping the membrane and carrier between. Buna-N O-ringsprovided watertight and gastight seals between the two half-chambers andthe membrane carrier.

A Luer manifold system allowed regulation of fluid flow into each halfchamber. Driving force for gas flow was provided by compressed gascylinders and for liquid flow by a peristaltic pump. Independent controlof flow rate into each chamber and pressure within each chamber wereachieved by varying the diameter of tubing draining the chamber. Thevolumetric flows of gases and liquid were measured by timing positivedisplacement of a liquid meniscus in calibrated pipets or syringes.

Phosphate-buffered saline (PBS) was stored in a reservoir and circulatedwith a peristaltic pump. After membrane flushing with carbon dioxide toprime the pores, both sides of the membranes were flushed with PBS, andthe inlet port of the permeate side sealed. The outlet port wasconnected to a calibrated syringe barrel, and an oil seal was placed onthe syringe barrel. Flow through the feed side of the chamber wasadjusted to produce transmembrane pressures of 1.00, 1.25, 1.50, 1.75and 2.00 psi.

Volumetric displacement of the PBS-air meniscus under the oil seal wastimed to calculate volume flow. Pressure-flow curves were created foreach pore size, and hydraulic permeabilities for PBS were calculated.

Example 10

This example describes membrane permselectivity testing with thenanopore membranes. Membrane arrays were examined under differentialinterference contrast light microscopy for defects. Defective membraneswithin an array were sealed with medical-grade epoxy (Loctite HM-100)and the array mounted in a custom-made Ussing chamber. Hydraulicpermeability to CO₂ and phosphate-buffered saline (PBS) of each membranewas compared to historical controls (CO₂) and first-principlesNavier-Stokes predictions (PBS). Membranes with measured hydraulicpermeability to PBS greater than 2× predicted were rejected. Theretentate side of the membrane was continuously perfused at a flow rateof 1 ml/min from a 100 ml reservoir of retentate, and the permeate sideof the membrane was wetted with 60 uL of PBS. Compressed air was used togenerate transmembrane pressures of 0, 1 or 2 psi as monitored by apressure transducer within the Ussing chamber (Entran, Inc). Themembrane was perfused for 200 hours with a solution of 4 mg/ml bovineserum albumin (Sigma, St. Louis) in PBS. The ultrafiltration volume wasmonitored either in a calibrated syringe barrel (Hamilton) capped withparafilm, or in a length of microbore tubing the tip of which wasimmersed in saline. Membranes were perfused with polydisperseFITC-labelled Ficoll 70 (50 ug/ml) prepared. Permeate was collected attimed intervals at zero membrane flux (diffusion-only data) and at 1 and2 psi driving pressure, corresponding to membrane fluxes ofapproximately 10⁻⁸ m/s. Feed and permeate samples were analyzed by gelpermeation chromatography with a Waters Ultrahydrogel 500 column on aWaters 600E system using a Waters 474 fluorescence detector. Sizecalibration of the column was performed with narrow-dispersion Ficollsamples of known size. Leaks were excluded by perfusing the membranewith 200 nm fluorescent beads, and examining the perfusate under afluorescent microscope.

A hollow-fiber dialyser (F200, Fresenius) was opened and individualhollow fibers potted in Luer fittings with medical-grade epoxy (LoctiteHM-100). Sample hollow fibers were perfused with Ficoll 70 by a circuitsimilar to that described above, and transmembrane pressure was set at 2psi. Ultrafiltrate samples were collected by gravity.

Sieving coefficients (fractional transmission) of three candidateproteins (ovalbumin, 45 kD, bovine serum albumin, 66 kD, andtransferrin, 76 kD) were individually tested with a 30 nm-wide slit poremembrane mounted in the custom-built apparatus (see, FIG. 7). Solutionsof each protein at 1 mg/ml concentration in pH 7.4 PBS without calciumwere prepared and protein concentration confirmed by Bradford assay.Next, a feed solution of bovine serum albumin was circulated on the feedside of the membrane, with continuous tangential flow at 1-2 ml/minacross the face of the membrane. Transmembrane pressures were adjustedto produce steady generation of permeate. After 100 μL of permeate wascollected, it was removed for analysis along with a simultaneous aliquotof feed solution.

The experiment was similarly repeated for ovalbumin and transferrin.Protein concentrations in feed and permeate were measured. Dead-spacevolume in the permeate chamber was measured by weighing the permeatevolume, and protein concentration in permeate was normalized to accountfor this dilution. Significant hindered transport was observed for allproteins in a size-dependent fashion (see, FIG. 11A) despite the factthat the membrane critical pore size was 3-5× than the moleculardiameter of the proteins. Solute transport of bovine serum albumin (BSA,Mw=66 kD) and Ficoll 70 (Mw=10-250 kD) was measured using a PEG-modifiedmembrane with 8 nm-wide pores mounted in the Fluid Flow Testing Station.A continuous flow of 50 μg/ml of FITC-labeled Ficoll 70 in PBS wasestablished across the FEED side, and pressurized with compressed air to2.0 psi to collect 80-110 μL of ultrafiltrate in the PERMEATE chamber.Afterwards, FEED and PERMEATE aliquot sample chromatograms were analyzedby gel permeation chromatography with a Waters Ultrahydrogel GPC columnon a 600E system. An identical procedure was followed with BSA, exceptthat the FEED and PERMEATE protein concentrations were measured byBradford assay. Sieving coefficients, σ, were calculated as the ratio ofPERMEATE/FEED concentration and compared to λ, which is the ratio of themolecular Stokes-Einstein diameter to nanopore size. The siliconnanoporous membrane displayed size-dependent rejection of solutes aspredicted by a steric hindrance model for rigid spheres (FIG. 11B).Specifically, BSA exhibited a sieving coefficient of 0.24, which isclose to the predicted value (see, e.g., Weinbaum, S., Lect. Math LifeSci., 1981. 14(119)). For Ficoll 70, transport was observed in excess ofpredictions for λ>0.8, as has been reported previously (see, e.g.,Rippe, C., et al., Kidney Int, 2006. 69(8): p. 1326; Venturoli, D. andB. Rippe, Am J Physiol Renal Physiol, 2005. 288(4): p. F605). Thepermselectivity data indicates, for example, that the nanopores are verydiscriminating based on molecular weight/size of the protein. Inadditional experiments, prototype silicon nanoporous membranes weremanufactured with highly uniform smooth walled pores (see, FIG. 10). Gastransport of carbon dioxide, nitrogen, and argon were consistent withtransition-regime and Knudsen flow. Membrane hydraulic permeabilitiesfor PBS closely matched first-principles predictions (see, FIG. 12).Hydraulic permeability of an array of six 1 mm² membranes was completelyunchanged by extended perfusion with bovine serum albumin (1.0 uL/min/6mm² at 2.0 PSI before and after BSA). Serial dilutions of retentate andpermeate revealed that fluorescent latex beads were observable at 10⁻⁵to 10⁻⁶ dilution of the retentate but were not observed in the permeateat any dilution. Prototype membranes displayed size-dependent rejectionof Ficolls (see, FIG. 13). The molecular weight cutoff of thePEG-modified silicon membrane, as estimated by the molecular weight atwhich the rejection coefficient is half-maximal, was around 18 kD,compared with less than 10 kD for the polysulfone hollow-fiber dialyser.

Example 11

This example describes the growth of renal tubule cells on nanoporousmembranes. Central to the goal of reducing need for replacement fluid isthe use of a cell bioreactor to reabsorb salt and water from theultrafiltrate stream while maintaining a barrier to reabsorption ofuremic toxins. Metabolic activity of renal epithelial cells areclinically important in stress states such as septic shock andmultisystem organ failure. The renal epithelial cells are typicallyderived from the cortex of donor kidneys that proved unsuitable fortransplant. Cells were seeded into the lumens of conventionalpolysulfone hollow-fiber dialyzer cartridges and grown to confluencebefore deployment in the extracorporeal circuit. The polymer surface ofthe hollow fibers was typically pretreated with Pronectin-L or murinecollagen to enhance cell attachment. Early single hollow-fiberexperiments demonstrated that solute transport by the cultured renalcortical epithelial cells was an active process, as blocking Na—K-ATPasewith oubain, and blocking sodium-dependent glucose transport withphlorizin altered apical-to-basal sodium and glucose concentrations.

Function of a miniaturized implantable device requires a miniaturizedbioreactor, which, in turn, is affected by epithelial cell attachment,maintenance of differentiated phenotype, and epithelial tight junctionintegrity. The suitability of silicon nanoporous membranes as scaffoldsfor tubule cell bioreactors has been demonstrated. Membranes with 40nm-wide slit pores were bathed in 30% hydrogen peroxide overnight toimprove surface wetting, followed by rinsing with phosphate bufferedsaline and adsorption of murine collagen IV at 5 μg/cm². Human corticaltubule cells were seeded at 5×10⁵ cells/cm² on nanoporous membranes aswell as control polyester cell cultures inserts (Corning Transwell, 0.4μm pore size) and grown to confluence in hormonally defined media.Transepithelial resistance was measured at intervals across nanoporousmembranes and polyester membranes using a standard instrument (EVOMX,World Precision Instruments). Cells appeared confluent after about sixdays, and the cells were fixed with 4% cold paraformaldehyde after atotal of 14d of growth. The cells were stained by indirectimmunofluorescence for a tight junction protein, ZO-1, a ciliaryprotein, acetylated tubulin, and a brush border enzyme, γ-glutamyltransferase, as well as a nuclear stain, 4′,6-Diamidino-2-phenylindole(DAPI). Cells grown on silicon nanoporous membranes and on polyestercontrols showed similarly intense staining for all three proteins,suggesting that the epithelial cells were able to achieve differentiatedphenotype similarly on standard tissue culture substrates and on thesilicon nanoporous membranes (see, FIG. 14, left). Transepithelialresistance was also highly similar between the two substrates,suggesting the formation of functional tight junctions (FIG. 14, right).These results show, for example, that nanoporous silicon membranes areviable as a scaffold for the cell bioreactor in a miniaturizedimplantable RAD.

Example 12

This example describes various alternative pore pattern designs andmembrane fabrication techniques.

In some embodiments, the present invention provides a targethemofiltration rate of 30 ml/min through arrays of sub-50 nm pores witha transmembrane pressure of 30 mmHg. These embodiments employ willhighly porous, yet mechanically robust, membranes. Preferably, thedesigns are configured to handle filtration, while exhibiting burststrengths of at least 300 mmHg.

Reliability analyses were performed on silicon nanoporous membranes ofidentical overall size, but several pore arrangements derived from thecurrent design (pore spacing of ˜2.5 μm). Stress fields on the membranesunder 5 psi (˜260 mmHg) transmembrane pressure were examined usingfinite element methods, and subsequently, reliability analyses wereconducted for each stress field using representative Weibull materialparameters (see, e.g., Jadaan, et al., Strength and Weibullcharacterization of polysilicon membranes for MEMS applications.Proceedings of the 30th International Conference & Exposition onAdvanced Ceramics & Composites, Cocoa Beach, Fla., 2006). Thesereliability analyses allowed for comparison of various nanoporearrangements relative to each other.

Table 1 depicts the stress fields and fracture probability of threemembrane designs. Design #A shows a baseline solid membrane without anypores, while Design #B presents a membrane design having 2.9% porositywherein the pores are distributed all over the membrane including theedge and center regions. The corresponding fracture probabilities (˜0%for Design #A and 55% for Design #B) indicate that current membranedesigns are fragile and fracture slightly above 3 psi, while the solidmembranes survive 100 psi transmembrane pressures. In contrast, poredistribution in Design #C has been optimized to avoid membrane edges andthe center region, which results in a very low fracture probability(˜0%).

The present invention provides a variety of different nanoporearrangement. In some embodiments, pore patterns are provided onmembranes in a rectangular configuration (e.g., at least 2:1) ratherthan a square (1:1 aspect ratio) to mitigate stress levels in the longdirection. It is contemplated that these modifications result in aprobability of failure that will be <1%. Hydraulic permeability of themembrane can be increased in two ways: (a) porosity can be increased to˜20% by decreasing the pore spacing from 2000 nm to 50 nm; and (b)channel conductance can be increased by decreasing membrane thickness˜4000 nm to 500 nm.

In some embodiments, the design for the hemofilter is a 1micrometer-thick membrane with arrays of 10-50 nm-wide slit pores with50 nm separation. This membrane geometry and nanopore arrangement iscontemplated to achieve 30 ml/min filtration rate with 30 mmHg oftransmembrane pressure within an overall membrane area of 0.1 m². Insome embodiments, a fabrication approach shown in FIG. 15 is used. FIG.15 shows the process via cross-sectional fabrication sequence: (a)process begins with deposition of a 1.0 μm low-stress nitride layer anda 1.0 μm polysilicon layer; (b) polysilicon layer is pattered usingnanoimprint lithography and RIE; (c) oxidation grows sacrificial oxidelayer on the polysilicon; RIE is used to remove oxide on top ofpolysilicon, and a 0.5 μm-thick polysilicon layer is deposited; (d)polysilicon is polished back on front side and a protective layer ofnitride and oxide is deposited on top; (e) cavities are etched from thebackside and the pores are opened via a hydrofluoric acid etch. Thenanopore arrangement can be of any type described herein. Preferably,the design decreases the pore spacing to as low as 50 nm by nanoimprintlithography (IMPRIO System) and RIE of the polysilicon. In someembodiments, the patterned wafers are then thermally oxidized to grow a10-50 nm-thick sacrificial oxide layer. The exact thickness of the oxidelayer will depend on the pore spacing.

In some embodiments, the fabrication protocols combine nanoimprintlithography, silicon-on-insulator (SOI) technology, and silicon waferbonding to produce robust nanoporous membranes supported by crossbars.In such embodiments, the pore size is not defined by oxidation, but by ananoimprint lithography and RIE step. Furthermore, the membranes areconstructed from one layer of single crystal silicon, therebyeliminating residual stresses associated with polysilicon.

Two distinct SOI wafers are used to achieve high-density and rigidmembranes (FIG. 16). FIG. 16 shows a cross-sectional fabricationsequence utilizing two SOI wafers: (a) bottom wafer SOI layer ispatterned and etched into a rectangular array using standard MEMSprocesses; (b) top wafer SOI layer is pattered using nanoimprintlithography and RIE; (c) the two wafers are fusion bonded together; (d)top substrate is removed, bottom substrate is patterned, and device isreleased by removing oxide from the backside and the pores are openedvia a hydrofluoric acid etch. The membrane geometry and layer thicknessvalues are representative values to convey process capabilities. In someembodiments, the first wafer, designated “BOT” for bottom wafer, is a400 μm-thick, double-side polished SOI wafer. The actual film thicknessmay be selected as desired. In some embodiments, the active layer is10-15 μm thick and the buried oxide (BOX) is 1.0 μm thick. The firststep is to pattern the SOI layer using standard photolithography anddeep reactive ion etching (DRIE). The SOI layer will be patterned intocrossbar patterns for optimal strength. The second wafer, designated“TOP” for top wafer, is a SmartCut™ SOI wafer with an active layer withthickness of 200-500 nm, while its BOX layer thickness is 200-300 nm.The silicon substrate is 400 μm thick.

The SOI layer of the top wafer can be patterned using nanoimprintlithography and RIE. Patterning of slit-patterns down to 10 nm-widths,for example, can be achieved via careful template fabrication (LeicaMicrosystems VB6UHR EWF) and process development (Molecular ImprintsIMPRIO-100, MRC-UTexas). The resulting pattern in the SOI layer of thetop wafer provides a high-density pattern comprising arrays of spaces assmall as, for example, 10 nm-wide (and up to 50 nm) with 50 nmseparation. In the next step, the two wafers are bonded together usingsilicon-to-silicon fusion bonding. Afterwards, the top wafer substrateis removed using XeF₂ etching and the bottom wafer substrate ispatterned using photolithography and DRIE. The final step of thefabrication process is to release the membranes by selectively etchingthe oxide layers in hydrofluoric acid.

In some embodiments, to avoid oxidation problems or limitations onlithography techniques, different pore designs are employed to achievesimilar results. For example, in some embodiments, the design increasesthe separation of slit pores to 100 nm, while decreasing membranethickness to 500 nm. It is contemplated that this design maintainsdesired hydraulic permeability. In some embodiments, the design approachinvolves patterning 50 nm-wide pores followed by thermal oxidation ofsidewalls.

In some embodiments, the target design for the hemofilter is a 0.5μm-thick membrane with arrays of <10 nm-wide slit pores with 100 nmseparation. In some embodiments, such membrane geometry and nanoporearrangement achieves 30 ml/min filtration rate with 30 mmHg oftransmembrane pressure within an overall membrane area of 0.1 m2. Thechoice of the 10 nm as maximum is derived from existing high-fluxpolysulfone hemodialysis membranes, which are rated with a mean poresize typically at 3-5 nm (see, e.g., Bowry, S. K., Int. J. Artif. Organs2002, 25(5), page 447). In some embodiments, a closer nanopore spacingin a 0.5 μm-thick membrane (see, FIG. 17) is achieved. In someembodiments, single crystal silicon is used instead of polysilicon, forthe first structural layer to avoid grain boundary effects for <10nm-thick oxide film growth.

Example 13

This example shows that membranes having slit shaped nanofabricatedpores offer enhanced selectivity in comparison to membranes having roundshaped nanofabricated pores. In experiments conducted during the courseof development of embodiments for the present invention, membraneshaving slit shaped nanofabricated pores in comparison to membraneshaving round shaped nanofabricated pores were shown to having enhancedclearance of middle-molecular weight toxins while still retainingalbumin.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entireties. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

We claim:
 1. An ultrafiltration system comprising: a) a membraneconsisting of a plurality of nanofabricated slit shaped pores having awidth less than 20 nanometers, wherein said plurality of nanofabricatedslit shaped pores are arranged in a rectangular configuration on saidmembrane, wherein said rectangular configuration is at least a 2:1arrangement of said nanofabricated slit shaped pores on said membrane,wherein said nanofabricated slit shaped pores are arranged in the centerof said membrane, wherein the distance between said nanofabricated slitshaped pores on said membrane is around 50 nm, wherein the thickness ofthe membrane is approximately 500 nm, wherein the membrane has aporosity around 20%; b) a housing containing said membrane; and c) afluid delivery passageway with a first end and a second end, said firstend positioned outside of said housing, said second end positioned todeliver fluid across said membrane.
 2. The system of claim 1, whereinsaid fluid is delivered across said membrane via perfusion pressure. 3.The system of claim 1, further comprising a pump configured to deliversaid fluid across said membrane.
 4. The system of claim 1, furthercomprising an actuator configured to decrease protein fouling of saidnanofabricated pores.
 5. The system of claim 1, further comprising acontrol component used to control/monitor extracellular fluid volume. 6.The system of claim 1, wherein the length of said nanofabricated poresis less than 50 μm.
 7. The system of claim 1, wherein said housingcomprises a coating, said coating being biocompatible for in vivo use.8. The system of claim 1, wherein said system further comprises one ormore electrodes positioned on or near said membrane such that anelectric field is generated in or near said pores.
 9. The system ofclaim 1, wherein said housing has a length and a width, said length ofsaid housing being less than 300 millimeters and said width of saidhousing being less than 300 millimeters.
 10. The system of claim 1,wherein said membrane has a surface coating selected from the groupconsisting of polyethylene glycol, oligosaccharide surfactant polymers,heparin, and hyaluronan.
 11. The system of claim 1, wherein the materialof said membrane is selected from the group consisting of silicon,polysilicon, silicon carbide, ultrananocrystalline diamond,diamond-like-carbond (DLC), silicon dioxide, PMMA, SU-8, PTFE, titanium,silica, silicon nitride, polytetrafluorethylene, polymethylmethacrylate,polystyrene, and silicone.
 12. An ultrafiltration system comprising: a)a membrane consisting of a plurality of nanofabricated slit shaped poreshaving a width less than 20 nanometers, wherein said plurality ofnanofabricated slit shaped pores are arranged in a rectangularconfiguration on said membrane, wherein said rectangular configurationis at least a 2:1 arrangement of said nanofabricated slit shaped poreson said membrane, wherein said nanofabricated slit shaped pores arearranged in the center of said membrane, wherein the distance betweensaid nanofabricated slit shaped pores on said membrane is around 50 nm,wherein the thickness of the membrane is approximately 500 nm, whereinthe membrane has a porosity around 20%, wherein said membrane has asurface coating selected from the group consisting of polyethyleneglycol, oligosaccharide surfactant polymers, heparin, and hyaluronan; b)a housing containing said membrane; and c) a fluid delivery passagewaywith a first end and a second end, said first end positioned outside ofsaid housing, said second end positioned to deliver fluid across saidmembrane.
 13. The system of claim 12, wherein said fluid is deliveredacross said membrane via perfusion pressure.
 14. The system of claim 12,further comprising a pump configured to deliver said fluid across saidmembrane.
 15. The system of claim 12, further comprising an actuatorconfigured to decrease protein fouling of said nanofabricated pores. 16.The system of claim 12, further comprising a control component used tocontrol/monitor extracellular fluid volume.
 17. The system of claim 12,said plurality of nanofabricated pores having a width less than 20nanometers.
 18. The system of claim 12, wherein the length of saidnanofabricated pores is less than 50 μm.
 19. The system of claim 12,wherein said housing comprises a coating, said coating beingbiocompatible for in vivo use.
 20. The system of claim 12, wherein saidsystem further comprises one or more electrodes positioned on or nearsaid membrane such that an electric field is generated in or near saidpores.
 21. The system of claim 12, wherein said housing has a length anda width, said length of said housing being less than 300 millimeters andsaid width of said housing being less than 300 millimeters.
 22. Thesystem of claim 12, wherein the material of said membrane is selectedfrom the group consisting of silicon, polysilicon, silicon carbide,ultrananocrystalline diamond, diamond-like-carbond (DLC), silicondioxide, PMMA, SU-8, PTFE, titanium, silica, silicon nitride,polytetrafluorethylene, polymethylmethacrylate, polystyrene, andsilicone.
 23. An ultrafiltration system comprising: a) a membraneconsisting of a plurality of nanofabricated slit shaped pores having awidth less than 20 nanometers, wherein said plurality of nanofabricatedslit shaped pores are arranged in a rectangular configuration on saidmembrane, wherein said rectangular configuration is at least a 2:1arrangement of said nanofabricated slit shaped pores on said membrane,wherein the distance between said nanofabricated slit shaped pores onsaid membrane is around 50 nm, wherein the thickness of the membrane isapproximately 500 nm, wherein the membrane has a porosity around 20%,wherein said nanofabricated slit shaped pores are arranged in the centerof said membrane; said membrane having a surface coating selected fromthe group consisting of polyethylene glycol, oligosaccharide surfactantpolymers, heparin, and hyaluronan; b) a housing containing said membraneand said electrode; and c) a fluid delivery passageway with a first endand a second end, said first end positioned outside of said housing,said second end positioned to deliver fluid across said membrane. 24.The system of claim 23, wherein said fluid is delivered across saidmembrane via perfusion pressure.
 25. The system of claim 23, furthercomprising a pump configured to deliver said fluid across said membrane.26. The system of claim 23, further comprising an actuator configured todecrease protein fouling of said nanofabricated pores.
 27. The system ofclaim 23, wherein the material of said membrane is selected from thegroup consisting of silicon, polysilicon, silicon carbide,ultrananocrystalline diamond, diamond-like-carbond (DLC), silicondioxide, PMMA, SU-8, PTFE, titanium, silica, silicon nitride,polytetrafluorethylene, polymethylmethacrylate, polystyrene, andsilicone.
 28. The system of claim 23, further comprising a controlcomponent used to control/monitor extracellular fluid volume.
 29. Thesystem of claim 23, wherein the length of said nanofabricated pores isless than 50 μm.
 30. The system of claim 23, wherein said housingcomprises a coating, said coating being biocompatible for in vivo use.31. The system of claim 23, wherein said system further comprises one ormore electrodes positioned on or near said membrane such that anelectric field is generated in or near said pores.
 32. The system ofclaim 23, wherein said housing has a length and a width, said length ofsaid housing being less than 300 millimeters and said width of saidhousing being less than 300 millimeters.
 33. An implantableultrafiltration device comprising: a) a membrane consisting of aplurality of nanofabricated slit shaped pores having a width less than20 nanometers, wherein said plurality of nanofabricated slit shapedpores are arranged in a rectangular configuration on said membrane,wherein said rectangular configuration is at least a 2:1 arrangement ofsaid nanofabricated slit shaped pores on said membrane, wherein saidnanofabricated slit shaped pores are arranged in the center of saidmembrane, wherein the distance between said nanofabricated slit shapedpores on said membrane is around 50 nm, wherein the thickness of themembrane is approximately 500 nm, wherein the membrane has a porosityaround 20%; said membrane having a surface coating selected from thegroup consisting of polyethylene glycol, oligosaccharide surfactantpolymers, heparin, and hyaluronan; b) a biocompatible housing containingsaid membrane; and c) a fluid delivery passageway with a first end and asecond end, said first end positioned outside of said housing, saidsecond end positioned to delivery fluid across said membrane.
 34. Thedevice of claim 33, wherein said fluid is delivered across said membranevia perfusion pressure.
 35. The device of claim 33, further comprising apump configured to deliver said fluid across said membrane.
 36. Thedevice of claim 33, further comprising an actuator configured todecrease protein fouling of said nanofabricated pores.
 37. The device ofclaim 33, further comprising a control component used to control/monitorextracellular fluid volume.
 38. The device of claim 33, wherein thematerial of said membrane is selected from the group consisting ofsilicon, polysilicon, silicon carbide, ultrananocrystalline diamond,diamond-like-carbond (DLC), silicon dioxide, PMMA, SU-8, PTFE, titanium,silica, silicon nitride, polytetrafluorethylene, polymethylmethacrylate,polystyrene, and silicone.
 39. The device of claim 33, wherein thelength of said nanofabricated pores is less than 50 μm.
 40. The deviceof claim 33, wherein said housing comprises a coating, said coatingbeing biocompatible for in vivo use.
 41. The device of claim 33, furthercomprising a population of cells attached to said membrane.
 42. Thedevice of claim 41, wherein said cells are selected from the groupconsisting of renal tubule cells, pancreatic cells, hepatic cells,thyroid cells, adrenal cells, parathyroid cells, pituitary cells,hypothalamic cells, gonadal cells, prokaryotic cells, duodenal cells,gastric cells, intestinal cells, muscle cells, fibroblast cells, andendothelial cells.
 43. The device of claim 33, wherein said housing hasphysical dimensions that permit said device to be used in a humansubject, in vivo.
 44. The device of claim 33, further comprising one ormore sensors associated with said membrane, wherein said sensor isconfigured to monitor filtration parameters.
 45. The device of claim 44,wherein said one or more sensors are selected from the group consistingof a pressure sensor configured to monitor transmembrane pressure, aproteins sensor configured to monitor protein leakage/membranebreakdown, an optical blood sensor configured to monitor membranerupture, and urea sensor configured to monitor urea clearance.